S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity

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 β-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.     

Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary

Background  

The processes by which eggs develop in the insect ovary are well characterized. Despite a large number of Drosophila mutants that cannot lay eggs, the way that the egg is moved along the reproductive tract from ovary to uterus is less well understood. We remedy this with an integrative study on the reproductive tract muscles (anatomy, innervation, contractions, aminergic modulation) in female flies.     

Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.     

Surfaces designed to control the projected area and shape of individual cells

Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on specific cell functions (e.g., proliferation, cytoskeletal organization). Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a nonadhesive interpenetrating polymer network [poly (acrylamide-co-ethylene glycol); P (AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100 microns 2 to 10,000 microns 2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.     

Meta-analysis identified the TNFA -308G > A promoter polymorphism as a risk factor for disease severity in patients with rheumatoid arthritis

Introduction

The goal of this study is to investigate whether the -308G > A promoter polymorphism in the tumor necrosis factor alpha (TNFA) gene is associated with disease severity and radiologic joint damage in a large cohort of patients with rheumatoid arthritis (RA).

Methods

A long-term observational early RA inception cohort (n = 208) with detailed information about disease activity and radiologic damage after 3, 6 and 9 years of disease was genotyped for the TNFA -308G > A promoter polymorphism (rs1800629). A longitudinal regression analysis was performed to assess the effect of genotype on RA disease severity and joint damage. Subsequently, a meta-analysis, including all publically available data, was performed to further test the association between joint erosions and the TNFA polymorphism. To learn more about the mechanism behind the effect of the polymorphism, RNA isolated from peripheral blood from RA patients (n = 66) was used for TNFA gene expression analysis by quantitative PCR.

Results

Longitudinal regression analysis with correction for gender and disease activity showed a significant difference in total joint damage between GG and GA+AA genotype groups (P = 0.002), which was stable over time. The meta-analysis, which included 2,053 patients, confirmed an association of the genetic variant with the development of erosions (odds ratio 0.78, 95% CI 0.62, 0.98). No significant differences in TNFA gene expression were observed for the different genotypes, confirming earlier findings in healthy individuals.

Conclusions

Our data confirm that the TNFA -308G > A promoter polymorphism is associated with joint damage in patients with RA. This is not mediated by differences in TNFA gene expression between genotypes.     

Alpha-melanocyte-stimulating hormone suppresses antigen-induced lymphocyte proliferation in humans independently of melanocortin 1 receptor gene status

Studies in mice indicate that alpha-melanocyte-stimulating hormone (alphaMSH) is immunosuppressive, but it is not known whether alphaMSH suppresses human immune responses to exogenous Ags. Human PBMCs, including monocytes, express the melanocortin 1 receptor (MC1R), and it is thought that the ability of alphaMSH to alter monocyte-costimulatory molecule expression and IL-10 release is mediated by this receptor. However, the MC1R gene is polymorphic, and certain MC1R variants compromise receptor signaling via cAMP, resulting in red hair and fair skin. Here, we have investigated whether alphaMSH can suppress Ag-induced lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. alphaMSH suppressed streptokinase-streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10(-13)-10(-11) M alphaMSH. Anti-IL-10 Abs failed to prevent suppression by alphaMSH, indicating that it was not due to MC1R-mediated IL-10 release by monocytes. Despite variability in the degree of suppression between subjects, similar degrees of alphaMSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R alleles. RT-PCR of streptokinase-streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macrophages, MC1R and MC3R expression by B lymphocytes, but no melanocortin receptor expression by T lymphocytes. In addition, alphaMSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas alphaMSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that alphaMSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lymphocytes, has immunosuppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by alphaMSH is independent of MC1R gene status.     

Putting the pH into phosphatidic acid signaling

Background

Camouflage patterns that hinder detection and/or recognition by antagonists are widely studied in both human and animal contexts. Patterns of contrasting stripes that purportedly degrade an observer's ability to judge the speed and direction of moving prey ('motion dazzle') are, however, rarely investigated. This is despite motion dazzle having been fundamental to the appearance of warships in both world wars and often postulated as the selective agent leading to repeated patterns on many animals (such as zebra and many fish, snake, and invertebrate species). Such patterns often appear conspicuous, suggesting that protection while moving by motion dazzle might impair camouflage when stationary. However, the relationship between motion dazzle and camouflage is unclear because disruptive camouflage relies on high-contrast markings. In this study, we used a computer game with human subjects detecting and capturing either moving or stationary targets with different patterns, in order to provide the first empirical exploration of the interaction of these two protective coloration mechanisms.

Results

Moving targets with stripes were caught significantly less often and missed more often than targets with camouflage patterns. However, when stationary, targets with camouflage markings were captured less often and caused more false detections than those with striped patterns, which were readily detected.

Conclusions

Our study provides the clearest evidence to date that some patterns inhibit the capture of moving targets, but that camouflage and motion dazzle are not complementary strategies. Therefore, the specific coloration that evolves in animals will depend on how the life history and ontogeny of each species influence the trade-off between the costs and benefits of motion dazzle and camouflage.