Protein 5A Research Articles

An integrated metabolomics and proteomics analysis reveals copper‑zinc alloy surface contact-mediated metabolic disruption, lipid peroxidation, and osmotic imbalance of Cryptocaryon irritans tomonts

Cryptocaryon irritans cause massive losses in the mariculture industry and are among the most threatening pathogens affecting teleost species. Copper‑zinc alloy (CZA) surface contact effectively kills C. irritans within minutes. Thus, this study employed integrated metabolomics and proteomics analysis to investigate the killing mechanism of CZA against C. irritans. Moreover, malondialdehyde contents, CuZn-SOD, and ATPase activities were evaluated. Proteomics analysis identified 142 differentially expressed proteins (DEPs), including 91 (59 upregulated, 32 downregulated), 92 (42 upregulated, 50 downregulated), and 43 (17 upregulated, 26 downregulated) DEPs at 0.5 h, 1 h, and 0.5 h vs 1 h of contact treatment with the CZA surface, respectively. Similarly, the metabolomic analysis identified 377 differentially expressed metabolites (DEMs). There were 237 (94 upregulated, 143 downregulated), 264 (112 upregulated, 152 downregulated), and 193 (101 upregulated, 92 downregulated) DEMs at 0.5 h, 1 h, and 0.5 h vs 1 h of contact treatment with the CZA surface, respectively. KEGG-based integrative analyses revealed that CZA exposure significantly enriched proteins and metabolites involved in oxidative phosphorylation, purine, pyrimidine, cysteine, and methionine metabolism. Notably, CZA exposure inhibited the TCA cycle, disrupting energy metabolism, as evidenced by downregulation of key TCA proteins, including citrate synthase and malate dehydrogenase, and metabolites like succinic and malic acids at both 0.5 h and 1 h of CZA treatment. Prolonged CZA exposure also affected protein metabolism, with significant downregulation of ribosomal protein S3a (RPS3a) and proteasome activity. Additionally, CZA surface contact downregulated key proteins involved in pyrimidine (DHFR-TS), purine (RRM1), and cysteine and methionine metabolism (AHCY), leading to disturbances in the methionine cycle, suppression of glutathione metabolism, and alterations in cellular redox status. Quantitative polymerase chain reaction (qPCR) analysis confirmed the upregulation of citrate synthase, proteasome subunit alpha type, and adenosylhomocysteinase with prolonged CZA exposure. Additionally, extended CZA exposure significantly decreased the MDA content due to the increased CuZn-SOD activity. Moreover, CZA exposure reduced Ca2+/Mg2+-ATPase and Na+/K+-ATPase activities. Altogether, CZA surface contact caused metabolic disruption, lipid peroxidation, and osmotic imbalance of C. irritans tomonts, highlighting the broad molecular impact of CZA on cellular processes.

Interaction of major facilitator superfamily domain containing 2A with the blood-brain barrier.

The functional and structural integrity of the blood-brain barrier is crucial in maintaining homeostasis in the brain microenvironment; however, the molecular mechanisms underlying the formation and function of the blood-brain barrier remain poorly understood. The major facilitator superfamily domain containing 2A has been identified as a key regulator of blood-brain barrier function. It plays a critical role in promoting and maintaining the formation and functional stability of the blood-brain barrier, in addition to the transport of lipids, such as docosahexaenoic acid, across the blood-brain barrier. Furthermore, an increasing number of studies have suggested that major facilitator superfamily domain containing 2A is involved in the molecular mechanisms of blood-brain barrier dysfunction in a variety of neurological diseases; however, little is known regarding the mechanisms by which major facilitator superfamily domain containing 2A affects the blood-brain barrier. This paper provides a comprehensive and systematic review of the close relationship between major facilitator superfamily domain containing 2A proteins and the blood-brain barrier, including their basic structures and functions, cross-linking between major facilitator superfamily domain containing 2A and the blood-brain barrier, and the in-depth studies on lipid transport and the regulation of blood-brain barrier permeability. This comprehensive systematic review contributes to an in-depth understanding of the important role of major facilitator superfamily domain containing 2A proteins in maintaining the structure and function of the blood-brain barrier and the research progress to date. This will not only help to elucidate the pathogenesis of neurological diseases, improve the accuracy of laboratory diagnosis, and optimize clinical treatment strategies, but it may also play an important role in prognostic monitoring. In addition, the effects of major facilitator superfamily domain containing 2A on blood-brain barrier leakage in various diseases and the research progress on cross-blood-brain barrier drug delivery are summarized. This review may contribute to the development of new approaches for the treatment of neurological diseases.

Structural basis for lipid transfer by the ATG2A-ATG9A complex.

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes. Autophagy-related proteins (ATGs) 2A and 9A have an essential role in autophagy by mediating lipid transfer and re-equilibration between membranes for autophagosome formation. Here we report the cryo-electron microscopy structures of human ATG2A in complex with WD-repeat protein interacting with phosphoinositides 4 (WIPI4) at 3.2 Å and the ATG2A-WIPI4-ATG9A complex at 7 Å global resolution. On the basis of molecular dynamics simulations, we propose a mechanism of lipid extraction from the donor membranes. Our analysis revealed 3:1 stoichiometry of the ATG9A-ATG2A complex, directly aligning the ATG9A lateral pore with ATG2A lipid transfer cavity, and an interaction of the ATG9A trimer with both the N-terminal and the C-terminal tip of rod-shaped ATG2A. Cryo-electron tomography of ATG2A liposome-binding states showed that ATG2A tethers lipid vesicles at different orientations. In summary, this study provides a molecular basis for the growth of the phagophore membrane and lends structural insights into spatially coupled lipid transport and re-equilibration during autophagosome formation.

Mutations in the WG and GW motifs of the three RNA silencing suppressors of grapevine fanleaf virus alter their systemic suppression ability and affect virus infectivity.

Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.

Characterization of humoral immune responses against SARS-CoV-2 accessory proteins in infected patients and mouse model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, encodes several accessory proteins that have been shown to play crucial roles in regulating the innate immune response. However, their expressions in infected cells and immunogenicity in infected humans and mice are still not fully understood. This study utilized various techniques such as luciferase immunoprecipitation system (LIPS), immunofluorescence ​assay (IFA), and western ​blot (WB) to detect accessory protein-specific antibodies in sera of COVID-19 patients. Specific antibodies to proteins 3a, 3b, 7b, 8 and 9c can be detected by LIPS, but only protein 3a antibody was detected by IFA or WB. Antibodies against proteins 3a and 7b were only detected in ICU patients, which may serve as a marker for predicting disease progression. Further, we investigated the expression of accessory proteins in SARS-CoV-2-infected cells and identified the expressions of proteins 3a, 6, 7a, 8, and 9b. We also analyzed their ability to induce antibodies in immunized mice and found that only proteins 3a, 6, 7a, 8, 9b and 9c were able to induce measurable antibody productions, but these antibodies lacked neutralizing activities and did not protect mice from SARS-CoV-2 infection. Our findings validate the expression of SARS-CoV-2 accessory proteins and elucidate their humoral immune response, providing a basis for protein detection assays and their role in pathogenesis.

The N-Terminal Region of Cucumber Mosaic Virus 2a Protein Is Involved in the Systemic Infection in Brassica juncea.

Brassica juncea belongs to the Brassicaceae family and is used as both an oilseed and vegetable crop. As only a few studies have reported on the cucumber mosaic virus (CMV) in B. juncea, we conducted this study to provide a basic understanding of the B. juncea and CMV interactions. B. juncea-infecting CMV (CMV-Co6) and non-infecting CMV (CMV-Rs1) were used. To identify the determinants of systemic infection in B. juncea, we first constructed infectious clones of CMV-Co6 and CMV-Rs1 and used them as pseudo-recombinants. RNA2 of CMV was identified as an important determinant in B. juncea because B. juncea were systemically infected with RNA2-containing pseudo-recombinants; CMV-Co6, R/6/R, and R/6/6 were systemically infected B. juncea. Subsequently, the amino acids of the 2a and 2b proteins were compared, and a chimeric clone was constructed. The chimeric virus R/6Rns/R6cp, containing the C-terminal region of the 2a protein of CMV-Rs1, still infects B. juncea. It is the 2a protein that determines the systemic CMV infection in B. juncea, suggesting that conserved 160G and 214A may play a role in systemic CMV infection in B. juncea.

Validation of reference gene stability for normalization of RT-qPCR in Phytophthora capsici Leonian during its interaction with Piper nigrum L.

The selection of stable reference genes for the normalization of reverse transcription quantitative real-time PCR (RT-qPCR) is generally overlooked despite being the crucial element in determining the accuracy of the relative expression of genes. In the present study, the stability of seven candidate reference genes: actin (act), α-tubulin (atub), β-tubulin (btub), translation elongation factor 1-α (ef1), elongation factor 2 (ef2), ubiquitin-conjugating enzyme (ubc) and 40S ribosomal protein S3A (ws21) in Phytophthora capsici has been validated. The validation was performed at six infection time points during its interaction with its susceptible host Piper nigrum, two developmental stages, and for the combined dataset. Four algorithms: geNorm, NormFinder, BestKeeper, and the ΔCt method were compared, and a comprehensive ranking order was produced using RefFinder. The overall analysis revealed that ef1, ws21, and ubc were identified as the three most stable genes in the combined dataset, ef1, ws21, and act were the most stable at the infection stages, and, ef1, btub, and ubc were most stable during the developmental stages. These findings were further corroborated by validating the P. capsici pathogenesis gene NPP1 expression. The findings are significant as this is the first study addressing the stability of reference genes for P. capsici–P. nigrum interaction studies.

Synthesis, antibacterial activity, and 3D-QASR studies of matrine-indole derivatives as potential antibiotics

Matrine and indole have antibacterial, anticancer, and other biological activities, in order to develop new antibiotics to solve the problem of multi-drug resistant bacteria. In this paper, we synthesized a series of 29 novel matrine derivatives as potential drug candidates by combining indole analogs and matrine. The antibacterial activity of these compounds was evaluated through minimum inhibitory concentration (MIC) assays against five bacterial strains (S. aureus, C. albicans, P. acnes, P. aeruginosa, and E. coli). The obtained results demonstrated promising antibacterial efficacy, particularly for compounds A20 and A18, which exhibited MICs.au values of 0.021 and 0.031 mg/ml, respectively, against S. aureus. Moreover, compounds A20 and A27 displayed remarkable MICc.al values of 2.806 and 4.519 mg/ml, respectively, against C. albicans, surpassing the performance of the clinical antibiotic penicillin G sodium (0.0368 mg/ml) and fluconazole (4.849 mg/ml). These findings underscore the significant bacteriostatic activity of the matrine derivatives. Furthermore, to gain a deeper understanding 3D-QSAR modeling was employed, revealing the critical influence of steric structure, charge distribution, hydrophobic interactions, and hydrogen bonding within the molecular structure on the bacteriostatic activity of the compounds. Additionally, molecular docking simulations shed light on the interaction between compound A20 and bacterial proteins, highlighting the involvement of hydrogen bonding, hydrophobic interactions, and π-π conjugation in the formation of stable complexes that inhibit the normal functioning of the proteins. This comprehensive analysis provided valuable insights into the antibacterial mechanism of the novel matrine derivatives, offering theoretical support for their potential application as antibiotics.

Comprehensive Elucidation of the Role of L and 2A Security Proteins on Cell Death during EMCV Infection.

The EMCV L and 2A proteins are virulence factors that counteract host cell defense mechanisms. Both L and 2A exhibit antiapoptotic properties, but the available data were obtained in different cell lines and under incomparable conditions. This study is aimed at checking the role of these proteins in the choice of cell death type in three different cell lines using three mutants of EMCV lacking functional L, 2A, and both proteins together. We have found that both L and 2A are non-essential for viral replication in HeLa, BHK, and RD cell lines, as evidenced by the viability of the virus in the absence of both functional proteins. L-deficient infection led to the apoptotic death of HeLa and RD cells, and the necrotic death of BHK cells. 2A-deficient infection induced apoptosis in BHK and RD cells. Infection of HeLa cells with the 2A-deficient mutant was finalized with exclusive caspase-dependent death with membrane permeabilization, morphologically similar to pyroptosis. We also demonstrated that inactivation of both proteins, along with caspase inhibition, delayed cell death progression. The results obtained demonstrate that proteins L and 2A play a critical role in choosing the path of cell death during infection, but the result of their influence depends on the properties of the host cells.

Walnut phosphatase 2A proteins interact with basic leucine zipper protein JrVIP1 to regulate osmotic stress response <i>via</i> calcium signaling

Walnut phosphatase 2A proteins interact with basic leucine zipper protein JrVIP1 to regulate osmotic stress response <i>via</i> calcium signaling

Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids.

Single-molecule antigen detection using nanopores offers a promising alternative for accurate virus testing to contain their transmission. However, the selective and efficient identification of small viral proteins directly in human biofluids remains a challenge. Here, we report a nanopore sensing strategy based on a customized DNA molecular probe that combines an aptamer and an antibody to enhance the single-molecule detection of mpox virus (MPXV) A29 protein, a small protein with an M.W. of ca. 14 kDa. The formation of the aptamer-target-antibody sandwich structures enables efficient identification of targets when translocating through the nanopore. This technique can accurately detect A29 protein with a limit of detection of ∼11 fM and can distinguish the MPXV A29 from vaccinia virus A27 protein (a difference of only four amino acids) and Varicella Zoster Virus (VZV) protein directly in biofluids. The simplicity, high selectivity, and sensitivity of this approach have the potential to contribute to the diagnosis of viruses in point-of-care settings.

The role of the encephalomyocarditis virus type 1 proteins L and 2A in the inhibition of the synthesis of cellular proteins and the accumulation of viral proteins during infection

Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A «security» proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery. To study the role of L and 2A «security» proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells. Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; Δ2A encoding a partially deleted 2A; Zfmut&Δ2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography. The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2α, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation. A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.

Genomic analysis of penicillin-binding proteins and recombination events in an emerging amoxicillin- and meropenem-resistant PMEN3 (Spain9V-3, ST156) variant in Taiwan and comparison with global descendants of this lineage.

From 2008 to 2020, the Taiwan National Notifiable Disease Surveillance System database demonstrated that the incidence of non-vaccine serotype 23A invasive pneumococcal disease (IPD) approximately doubled. In this study, 276 non-repetitive pneumococcal clinical isolates were collected from two medical centers in Taiwan between 2019 and 2021. Of these 267 pneumococci, 60 were serotype 23A. Among them, 50 (83%) of serotype 23A isolates belonged to the sequence type (ST) 166 variant of the Spain9V-3 clone. Pneumococcal 23A-ST166 isolates were collected to assess their evolutionary relationships using whole-genome sequencing. All 23A-ST166 isolates were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299, the newly identified PBP2x-299 in Taiwan. Transformation of the pbp1a, pbp2b, and pbp2x alleles into the β-lactam-susceptible R6 strain revealed that PBP2x-299 and PBP2b-11 increased the MIC of ceftriaxone and meropenem by 16-fold, respectively. Prediction analysis of recombination sites in PMEN3 descendants (23A-ST166 in Taiwan, 35B-ST156 in the United States, and 11A-ST838/ST6521 in Europe) showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displayed an evolutionary capacity for global dissemination and persistence, increasing IPD incidence, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases, and contributing to high antibiotic resistance.A clonal shift with a highly β-lactam-resistant non-vaccine serotype 23A, from ST338 to ST166, increased in Taiwan. ST166 is a single-locus variant of the Spain9V-3 clone, which is also called the PMEN3 lineage. All 23A-ST166 isolates, in this study, were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299. PBP2x-299 and PBP2b-11 contributed to the increasing MIC of ceftriaxone and meropenem, respectively. Prediction analysis of recombination sites in PMEN3 descendants showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displays the evolutionary capacity for dissemination, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases and contributing to high antibiotic resistance.

An unexpected, pH-sensitive step of the enterovirus D68 lifecycle.

Enterovirus D68 (EV-D68) contributes significantly to pathogen-induced respiratory illnesses and severe neurological disorders like acute flaccid myelitis. We lack EV-D68 preventive measures, and knowledge of its molecular and cellular biology is incomplete. Multiple studies have highlighted the role of membrane compartments and autophagy during picornavirus multiplication. Galitska et al. found that EV-D68 also exploits cellular autophagic compartments and relies on autophagic machinery as pro-viral factors (G. Galitska, A. Jassey, M. A. Wagner, N. Pollack, et al., mBio e02141-23, 2023, https://doi.org/10.1128/mbio.02141-23). Surprisingly, failure of the autophagic compartment to acidify early during EV-D68 infection causes a delay in RNA synthesis that has not been reported for other enteroviruses. This delay appears to reflect the inability of viral proteins 2B and 3A to engage membranes stably, leading to their degradation in the cytoplasm. Observations like this underscore the importance of studying individual members of the virus genus. It will be interesting to understand how this phenomenon connects to EV-D68 pathogenesis, if at all.

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.

Grapevine Fanleaf Virus RNA1-Encoded Proteins 1A and 1BHel Suppress RNA Silencing.

Grapevine fanleaf virus (GFLV) (genus Nepovirus, family Secoviridae) causes fanleaf degeneration, one of the most damaging viral diseases of grapevines. Despite substantial advances at deciphering GFLV-host interactions, how this virus overcomes the host antiviral pathways of RNA silencing is poorly understood. In this study, we identified viral suppressors of RNA silencing (VSRs) encoded by GFLV, using fluorescence assays, and tested their capacity at modifying host gene expression in transgenic Nicotiana benthamiana expressing the enhanced green fluorescent protein gene (EGFP). Results revealed that GFLV RNA1-encoded protein 1A, for which a function had yet to be assigned, and protein 1BHel, a putative helicase, reverse systemic RNA silencing either individually or as a fused form (1ABHel) predicted as an intermediary product of RNA1 polyprotein proteolytic processing. The GFLV VSRs differentially altered the expression of plant host genes involved in RNA silencing, as shown by reverse transcription-quantitative PCR. In a co-infiltration assay with an EGFP hairpin construct, protein 1A upregulated NbDCL2, NbDCL4, and NbRDR6, and proteins 1BHel and 1A+1BHel upregulated NbDCL2, NbDCL4, NbAGO1, NbAGO2, and NbRDR6, while protein 1ABHel upregulated NbAGO1 and NbRDR6. In a reversal of systemic silencing assay, protein 1A upregulated NbDCL2 and NbAGO2 and protein 1ABHel upregulated NbDCL2, NbDCL4, and NbAGO1. This is the first report of VSRs encoded by a nepovirus RNA1 and of two VSRs that act either individually or as a predicted fused form to counteract the systemic antiviral host defense, suggesting that GFLV might devise a unique counterdefense strategy to interfere with various steps of the plant antiviral RNA silencing pathways during infection. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

SARS-CoV-2 ORF8 accessory protein is a virulence factor.

The relevance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF8 in the pathogenesis of COVID-19 is unclear. Virus natural isolates with deletions in ORF8 were associated with wild milder disease, suggesting that ORF8 might contribute to SARS-CoV-2 virulence. This manuscript shows that ORF8 is involved in inflammation and in the activation of macrophages in two experimental systems: humanized K18-hACE2 transgenic mice and organoid-derived human airway cells. These results identify ORF8 protein as a potential target for COVID-19 therapies.

Sodium Tanshinone IIA Sulfonate Ameliorates Oxygen-glucose Deprivation/Reoxygenation-induced Neuronal Injury via Protection of Mitochondria and Promotion of Autophagy.

Sodium tanshinone IIA sulfonate (STS) has shown significant clinical therapeutic effects in cerebral ischemic stroke (CIS), but the molecular mechanisms of neuroprotection remain partially known. The purpose of this study was to explore whether STS plays a protective role in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by regulating microglia autophagy and inflammatory activity. Co-cultured microglia and neurons were subjected to OGD/R injury, an in vitro model of ischemia/reperfusion (I/R) injury with or without STS treatment. Expression of protein phosphatase 2A (PP2A) and autophagy-associated proteins Beclin 1, autophagy related 5 (ATG5), and p62 in microglia was determined by Western blotting. Autophagic flux in microglia was observed with confocal laser scanning microscopy. Neuronal apoptosis was measured by flow cytometric and TUNEL assays. Neuronal mitochondrial function was determined via assessments of reactive oxygen species generation and mitochondrial membrane potential integrity. STS treatment markedly induced PP2A expression in microglia. Forced overexpression of PP2A increased levels of Beclin 1 and ATG5, decreased the p62 protein level, and induced autophagic flux. Silencing of PP2A or administration of 3-methyladenine inhibited autophagy and decreased the production of anti-inflammatory factors (IL-10, TGF-β and BDNF) and induced the release of proinflammatory cytokines (IL-1β, IL-2 and TNF-α) by STS-treated microglia, thereby inducing mitochondrial dysfunction and apoptosis of STS-treated neurons. STS exerts protection against neuron injury, and the PP2A gene plays a crucial role in improving mitochondrial function and inhibiting neuronal apoptosis by regulating autophagy and inflammation in microglia.

Reviewing the Regulators of COL1A1.

The collagen family contains 28 proteins, predominantly expressed in the extracellular matrix (ECM) and characterized by a triple-helix structure. Collagens undergo several maturation steps, including post-translational modifications (PTMs) and cross-linking. These proteins are associated with multiple diseases, the most pronounced of which are fibrosis and bone diseases. This review focuses on the most abundant ECM protein highly implicated in disease, type I collagen (collagen I), in particular on its predominant chain collagen type I alpha 1 (COLα1 (I)). An overview of the regulators of COLα1 (I) and COLα1 (I) interactors is presented. Manuscripts were retrieved searching PubMed, using specific keywords related to COLα1 (I). COL1A1 regulators at the epigenetic, transcriptional, post-transcriptional and post-translational levels include DNA Methyl Transferases (DNMTs), Tumour Growth Factor β (TGFβ), Terminal Nucleotidyltransferase 5A (TENT5A) and Bone Morphogenic Protein 1 (BMP1), respectively. COLα1 (I) interacts with a variety of cell receptors including integrinβ, Endo180 and Discoidin Domain Receptors (DDRs). Collectively, even though multiple factors have been identified in association to COLα1 (I) function, the implicated pathways frequently remain unclear, underscoring the need for a more spherical analysis considering all molecular levels simultaneously.

Nonstructural proteins 2B and 4A of Tembusu virus induce complete autophagy to promote viral multiplication in vitro

Tembusu virus (TMUV) is an emerging flavivirus that has broken out in different regions of China. TMUV infection has been reported to induce autophagy in duck embryo fibroblast cells. However, the molecular mechanisms underlying this autophagy induction remain unclear. Here, we explored the interactions between autophagy and TMUV and the effects of the structural and nonstructural proteins of TMUV on autophagy in vitro. Among our results, TMUV infection enhanced autophagy to facilitate viral replication in HEK293T cells. After pharmacologically inducing autophagy with rapamycin (Rapa), the replication of TMUV increased by a maximum of 14-fold compared with the control group. To determine which TMUV protein primarily induced autophagy, cells were transfected with two structural proteins and seven nonstructural proteins of TMUV. Western blotting showed that nonstructural proteins 2B (NS2B) and 4 A (NS4A) of TMUV significantly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3) from LC3-I to LC3-II in HEK293T cells. In addition, through immunofluorescence assays, we found that NS2B and NS4A significantly increased the punctate fluorescence of GFP-LC3-II. Furthermore, we found that both NS2B and NS4A interacted with polyubiquitin-binding protein sequestosome 1 (SQSTM1/p62) in a coimmunoprecipitation assay. Moreover, the autophagic degradation of p62 and LC3 mediated by NS2B or NS4A was inhibited by treatment with the autophagic flux inhibitor chloroquine (CQ). These results confirmed the vital effects of NS2B and NS4A in TMUV-induced complete autophagy and clarified the importance of complete autophagy for viral replication, providing novel insight into the relationship between TMUV and autophagy.