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1.
Int J Mol Sci ; 23(9)2022 May 03.
Article in English | MEDLINE | ID: covidwho-1820295

ABSTRACT

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein substrates. The crystal structure of human DHHC20 (hDHHC20), an enzyme involved in the acylation of S-protein of SARS-CoV-2, revealed that the acyl chain may be inserted into a hydrophobic cavity formed by four transmembrane (TM) α-helices. To test this model, we used molecular dynamics of membrane-embedded hDHHC20 and its mutants either in the absence or presence of various acyl-CoAs. We found that among a range of acyl chain lengths probed only C16 adopts a conformation suitable for hDHHC20 autoacylation. This specificity is altered if the small or bulky residues at the cavity's ceiling are exchanged, e.g., the V185G mutant obtains strong preferences for binding C18. Surprisingly, an unusual hydrophilic ridge was found in TM helix 4 of hDHHC20, and the responsive hydrophilic patch supposedly involved in association was found in the 3D model of the S-protein TM-domain trimer. Finally, the exchange of critical Thr and Ser residues in the spike led to a significant decrease in its S-acylation. Our data allow further development of peptide/lipid-based inhibitors of hDHHC20 that might impede replication of Corona- and other enveloped viruses.


Subject(s)
Acyltransferases , COVID-19 , Acyl Coenzyme A/metabolism , Acylation , Acyltransferases/chemistry , Acyltransferases/metabolism , Fatty Acids/chemistry , Fatty Acids/metabolism , Humans , Molecular Dynamics Simulation , SARS-CoV-2 , Substrate Specificity/physiology
2.
mBio ; 12(5): e0234221, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1494971

ABSTRACT

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.


Subject(s)
Acyltransferases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Alphacoronavirus/pathogenicity , Nerve Tissue Proteins/metabolism , Acyltransferases/genetics , Adaptor Proteins, Signal Transducing/genetics , Alphacoronavirus/drug effects , Animals , COVID-19/metabolism , HeLa Cells , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Nerve Tissue Proteins/genetics , Palmitates/pharmacology , SARS Virus/drug effects , SARS Virus/pathogenicity , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Swine
3.
Dev Cell ; 56(20): 2790-2807.e8, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1446559

ABSTRACT

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.


Subject(s)
Acylation/physiology , COVID-19/drug therapy , Membrane Lipids/metabolism , SARS-CoV-2/pathogenicity , Acyltransferases/metabolism , Golgi Apparatus/metabolism , Golgi Apparatus/virology , Humans , Virus Assembly/physiology
4.
J Biol Chem ; 297(4): 101112, 2021 10.
Article in English | MEDLINE | ID: covidwho-1364203

ABSTRACT

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of ß-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.


Subject(s)
COVID-19/pathology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Acylation , Acyltransferases/genetics , Acyltransferases/metabolism , Amino Acid Sequence , COVID-19/virology , Cysteine/metabolism , HEK293 Cells , Humans , Lipoylation , Mutagenesis, Site-Directed , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
5.
Plant J ; 107(5): 1299-1319, 2021 09.
Article in English | MEDLINE | ID: covidwho-1282039

ABSTRACT

Caffeoylquinic acids (CQAs) are specialized plant metabolites we encounter in our daily life. Humans consume CQAs in mg-to-gram quantities through dietary consumption of plant products. CQAs are considered beneficial for human health, mainly due to their anti-inflammatory and antioxidant properties. Recently, new biosynthetic pathways via a peroxidase-type p-coumaric acid 3-hydroxylase enzyme were discovered. More recently, a new GDSL lipase-like enzyme able to transform monoCQAs into diCQA was identified in Ipomoea batatas. CQAs were recently linked to memory improvement; they seem to be strong indirect antioxidants via Nrf2 activation. However, there is a prevalent confusion in the designation and nomenclature of different CQA isomers. Such inconsistencies are critical and complicate bioactivity assessment since different isomers differ in bioactivity and potency. A detailed explanation regarding the origin of such confusion is provided, and a recommendation to unify nomenclature is suggested. Furthermore, for studies on CQA bioactivity, plant-based laboratory animal diets contain CQAs, which makes it difficult to include proper control groups for comparison. Therefore, a synthetic diet free of CQAs is advised to avoid interferences since some CQAs may produce bioactivity even at nanomolar levels. Biotransformation of CQAs by gut microbiota, the discovery of new enzymatic biosynthetic and metabolic pathways, dietary assessment, and assessment of biological properties with potential for drug development are areas of active, ongoing research. This review is focused on the chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity recently reported for mono-, di-, tri-, and tetraCQAs.


Subject(s)
Anti-Inflammatory Agents/chemistry , Antioxidants/chemistry , Cognitive Dysfunction/prevention & control , Neuroprotective Agents/chemistry , Phytochemicals/chemistry , Plants, Medicinal/chemistry , Quinic Acid/analogs & derivatives , Acyltransferases/genetics , Acyltransferases/metabolism , Animals , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Antioxidants/metabolism , Antioxidants/pharmacology , Biosynthetic Pathways , Brachypodium/enzymology , Dietary Supplements , Humans , Ipomoea batatas/enzymology , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Neuroprotective Agents/metabolism , Neuroprotective Agents/pharmacology , Phytochemicals/metabolism , Phytochemicals/pharmacology , Plant Proteins/genetics , Plant Proteins/metabolism , Quinic Acid/chemistry , Quinic Acid/metabolism , Quinic Acid/pharmacology , Terminology as Topic
6.
Bioorg Chem ; 112: 104925, 2021 07.
Article in English | MEDLINE | ID: covidwho-1198631

ABSTRACT

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.


Subject(s)
Acyltransferases/metabolism , Anti-Infective Agents/chemistry , Bacterial Proteins/metabolism , Resorcinols/chemistry , Acyltransferases/antagonists & inhibitors , Acyltransferases/classification , Acyltransferases/genetics , Anti-Infective Agents/isolation & purification , Anti-Infective Agents/metabolism , Anti-Infective Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/classification , Bacterial Proteins/genetics , COVID-19/pathology , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Drug Evaluation, Preclinical , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Molecular Conformation , Phylogeny , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Resorcinols/isolation & purification , Resorcinols/metabolism , Resorcinols/pharmacology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Streptomyces/enzymology , Tandem Mass Spectrometry
7.
Expert Opin Drug Discov ; 15(2): 159-177, 2020 02.
Article in English | MEDLINE | ID: covidwho-825219

ABSTRACT

Introduction: S-acylation is the attachment of fatty acids not only to cysteines of cellular, but also of viral proteins. The modification is often crucial for the protein´s function and hence for virus replication. Transfer of fatty acids is mediated by one or several of the 23 members of the ZDHHC family of proteins. Since their genes are linked to various human diseases, they represent drug targets.Areas covered: The authors explore whether targeting acylation of viral proteins might be a strategy to combat viral diseases. Many human pathogens contain S-acylated proteins; the ZDHHCs involved in their acylation are currently identified. Based on the 3D structure of two ZDHHCs, the regulation and the biochemistry of the palmitolyation reaction and the lipid and protein substrate specificities are discussed. The authors then speculate how ZDHHCs might recognize S-acylated membrane proteins of Influenza virus.Expert opinion: Although many viral diseases can now be treated, the available drugs bind to viral proteins that rapidly mutate and become resistant. To develop inhibitors for the genetically more stable cellular ZDHHCs, their binding sites for viral substrates need to be identified. If only a few cellular proteins are recognized by the same binding site, development of specific inhibitors may have therapeutic potential.


Subject(s)
Acyltransferases/metabolism , Antiviral Agents/pharmacology , Virus Diseases/drug therapy , Acylation/physiology , Animals , Binding Sites , Drug Development , Fatty Acids/metabolism , Humans , Lipoylation/physiology , Viral Proteins/metabolism , Virus Diseases/enzymology , Virus Diseases/virology
8.
Nature ; 585(7826): 614-619, 2020 09.
Article in English | MEDLINE | ID: covidwho-744380

ABSTRACT

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Subject(s)
Alkaloids/biosynthesis , Alkaloids/supply & distribution , Hyoscyamine/biosynthesis , Saccharomyces cerevisiae/metabolism , Scopolamine/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Animals , Atropa belladonna/enzymology , Atropine Derivatives/metabolism , Biological Transport , Datura/enzymology , Glucosides/biosynthesis , Glucosides/metabolism , Hyoscyamine/supply & distribution , Lactates/metabolism , Ligases/genetics , Ligases/metabolism , Models, Molecular , Nervous System Diseases/drug therapy , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Engineering , Saccharomyces cerevisiae/genetics , Scopolamine/supply & distribution , Vacuoles/metabolism
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