Translation landscape of SARS-CoV-2 noncanonical subgenomic RNAs

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a positive-stranded RNA genome. Current proteomic studies of SARS-CoV-2 mainly focus on the proteins encoded by its genomic RNA (gRNA) or canonical subgenomic RNAs (sgRNAs). Here, we systematically investigated the translation landscape of SARS-CoV-2, especially its noncanonical sgRNAs. We first constructed a strict pipeline, named vipep, for identifying reliable peptides derived from RNA viruses using RNA-seq and mass spectrometry data. We applied vipep to analyze 24 sets of mass spectrometry data related to SARS-CoV-2 infection. In addition to known canonical proteins, we identified many noncanonical sgRNA-derived peptides, which stably increase after viral infection. Furthermore, we explored the potential functions of those proteins encoded by noncanonical sgRNAs and found that they can bind to viral RNAs and may have immunogenic activity. The generalized vipep pipeline is applicable to any RNA viruses and these results have expanded the SARSCoV-2 translation map, providing new insights for understanding the functions of SARS-CoV-2 sgRNAs.     

Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins

Protein lysine acetylation is a reversible and highly regulated post‐translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology.     

Comparative acetylomic analysis reveals differentially acetylated proteins regulating fungal metabolism in hypovirus-infected chestnut blight fungus

Cryphonectria parasitica, the chestnut blight fungus, and hypoviruses are excellent models for examining fungal pathogenesis and virus–host interactions. Increasing evidence suggests that lysine acetylation plays a regulatory role in cell processes and signalling. To understand protein regulation in C. parasitica by hypoviruses at the level of posttranslational modification, a label-free comparative acetylome analysis was performed in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Using enrichment of acetyl-peptides with a specific anti-acetyl-lysine antibody, followed by high accuracy liquid chromatography–tandem mass spectrometry analysis, 638 lysine acetylation sites were identified on 616 peptides, corresponding to 325 unique proteins. Further analysis revealed that 80 of 325 proteins were differentially acetylated between C. parasitica strain EP155 and EP155/CHV1-EP713, with 43 and 37 characterized as up- and down-regulated, respectively. Moreover, 75 and 65 distinct acetylated proteins were found in EP155 and EP155/CHV1-EP713, respectively. Bioinformatics analysis revealed that the differentially acetylated proteins were involved in various biological processes and were particularly enriched in metabolic processes. Differences in acetylation in C. parasitica citrate synthase, a key enzyme in the tricarboxylic acid cycle, were further validated by immunoprecipitation and western blotting. Site-specific mutagenesis and biochemical studies demonstrated that the acetylation of lysine-55 plays a vital role in the regulation of the enzymatic activity of C. parasitica citrate synthase in vitro and in vivo. These findings provide a valuable resource for the functional analysis of lysine acetylation in C. parasitica, as well as improving our understanding of fungal protein regulation by hypoviruses from a protein acetylation perspective.     

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm,Bombyx mori

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H₂O₂. In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H₂O₂-induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.     

Smoking enhances the expression of angiotensin-converting enzyme 2 involved in the efficiency of severe acute respiratory syndrome coronavirus 2 infection

Smoking is one of the risk factors most closely related to the severity of coronavirus disease 2019 (COVID-19). However, the relationship between smoking history and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and nonsmokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of nonsmokers and ex-smokers with a long history of nonsmoking (cessation period longer than 6 months). We also showed that the efficiency of SARS-CoV-2 infection was enhanced in a manner dependent on the angiotensin-converting enzyme 2 (ACE2) expression level. Using RNA-seq analysis on the lungs of smokers, we identified that the expression of inflammatory signaling genes was correlated with ACE2 expression. Notably, with increasing duration of smoking cessation among ex-smokers, not only ACE2 expression level but also the expression levels of inflammatory signaling genes decreased. These results indicated that smoking enhances the expression levels of ACE2 and inflammatory signaling genes. Our data suggest that the efficiency of SARS-CoV-2 infection is enhanced by smoking-mediated upregulation of ACE2 expression level.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite‐specific functions during Plasmodium falciparum intraerythrocytic development

Lysine acetylation has emerged as a major post‐translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine‐acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium‐specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.     

Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus

Recent analysis of prokaryotic N^ε‐lysine‐acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N^ε‐lysine‐acetylated proteome of gram‐positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl‐lysine‐specific antibodies followed by LC‐MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.     

System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.     

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration. Supplementary InformationThe online version contains supplementary material available at 10.1007/s13238-022-00905-7.     

Structure of Sir2Tm bound to a propionylated peptide

Lysine propionylation is a recently identified post‐translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyl‐lysine is slightly reduced to acetyl‐lysine. These results serve to broaden our understanding of the newly identified propionyl‐lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.     

Prospects of Coffee Leaf against SARS-CoV-2 Infection

In the current climate, many countries are in dire need of effective preventive methods to curb the Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) pandemic. The purpose of this research is to screen and explore natural plant extracts that have the potential to against SARS-CoV-2 and provide alternative options for SARS-CoV-2 prevention and hand sanitizer or spray-like disinfectants. We first used Spike-ACE2 ELISA and TMPRSS2 fluorescence resonance energy transfer (FRET) assays to screen extracts from agricultural by-products from Taiwan with the potential to impede SARS-CoV-2 infection. Next, the SARS-CoV-2 pseudo-particles (Vpp) infection assay was tested to validate the effectiveness. We identified an extract from coffee leaf (Coffea Arabica), a natural plant that effectively inhibited wild-type SARS-CoV-2, and five Variants of Concern (Alpha, Beta, Gamma, Delta, and Omicron strain) from entering host cells. In an attempt to apply coffee leaf extract for hand sanitizer or spray-like disinfectants, we designed a skin-like gelatin membrane experiment. We showed that the high concentration of coffee leaf extract on the skin surface could block SARS-CoV-2 into cells more potently than 75% Ethanol, a standard disinfectant to inactivate SARS-CoV-2. Finally, LC-HRMS analysis was used to identify compounds such as caffeine, chlorogenic acid (CGA), quinic acid, and mangiferin that are associated with an anti-SARS-CoV-2 activity. Our results demonstrated that coffee leaf extract, an agricultural by-product effectively inhibits SARS-CoV-2 Vpp infection through an ACE2-dependent mechanism and may be utilized to develop products against SARS-CoV-2 infection.