HIV‐1 infection of CD4 T cells impairs antigen‐specific B cell function

Failures to produce neutralizing antibodies upon HIV‐1 infection result in part from B‐cell dysfunction due to unspecific B‐cell activation. How HIV‐1 affects antigen‐specific B‐cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV‐1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B‐cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T‐cell–B‐cell immune synapse. This interference reduced B‐cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV‐mediated dysfunction of antigen‐specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.     

Interactomes of SARS‐CoV‐2 and human coronaviruses reveal host factors potentially affecting pathogenesis

Host–virus protein–protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity‐labeling strategies and identified 437 human proteins as the high‐confidence interacting proteins. Further characterization of these interactions and comparison to other large‐scale study of cellular responses to SARS‐CoV‐2 infection elucidated how distinct SARS‐CoV‐2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID‐19. The interactomes of two key SARS‐CoV‐2‐encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein–protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS‐CoV‐2 provides valuable resources for the understanding and treating of this disease.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth

The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P₃) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P₃ levels in macrophages. The modulation of cellular PI(3,4,5)P₃/PIP₂ ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.     

Memory IgM protects endogenous insulin from autoimmune destruction

The enormous diversity of antibody specificities is generated by random rearrangement of immunoglobulin gene segments and is important for general protection against pathogens. Since random rearrangement harbors the risk of producing self‐destructive antibodies, it is assumed that autoreactive antibody specificities are removed during early B‐cell development leading to a peripheral compartment devoid of autoreactivity. Here, we immunized wild‐type mice with insulin as a common self‐antigen and monitored diabetes symptoms as a measure for autoimmune disease. Our results show that autoreactive anti‐insulin IgM and IgG antibodies associated with autoimmune diabetes can readily be generated in wild‐type animals. Surprisingly, recall immunizations induced increased titers of high‐affinity insulin‐specific IgM, which prevented autoimmune diabetes. We refer to this phenomenon as adaptive tolerance, in which high‐affinity memory IgM prevents autoimmune destruction by competing with self‐destructive antibodies. Together, this study suggests that B‐cell tolerance is not defined by the absolute elimination of autoreactive specificities, as harmful autoantibody responses can be generated in wild‐type animals. In contrast, inducible generation of autoantigen‐specific affinity‐matured IgM acts as a protective mechanism preventing self‐destruction.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

Integrative analysis provides multi‐omics evidence for the pathogenesis of placenta percreta

Pernicious placenta previa with placenta percreta (PP) is a catastrophic condition during pregnancy. However, the underlying pathogenesis remains unclear. In the present study, the placental tissues of normal cases and PP tissues of pernicious placenta previa cases were collected to determine the expression profile of protein‐coding genes, miRNAs, and lncRNAs through sequencing. Weighted gene co‐expression network analysis (WGCNA), accompanied by miRNA target prediction and correlation analysis, were employed to select potential hub protein‐coding genes and lncRNAs. The expression levels of selected protein‐coding genes, Wnt5A and MAPK13, were determined by quantitative PCR and immunohistochemical staining, and lncRNA PTCHD1‐AS and PAPPA‐AS1 expression levels were determined by quantitative PCR and fluorescence in situ hybridization. The results indicated that 790 protein‐coding genes, 382 miRNAs, and 541 lncRNAs were dysregulated in PP tissues, compared with normal tissues. WGCNA identified coding genes in the module (ME) black and ME turquoise modules that may be involved in the pathogenesis of PP. The selected potential hub protein‐coding genes, Wnt5A and MAPK13, were down‐regulated in PP tissues, and their expression levels were positively correlated with the expression levels of PTCHD1‐AS and PAPPA‐AS1. Further analysis demonstrated that PTCHD1‐AS and PAPPA‐AS1 regulated Wnt5A and MAPK13 expression by interacting with specific miRNAs. Collectively, our results provided multi‐omics data to better understand the pathogenesis of PP and help identify predictive biomarkers and therapeutic targets for PP.     

SARS‐CoV‐2 sensing by RIG‐I and MDA5 links epithelial infection to macrophage inflammation

SARS‐CoV‐2 infection causes broad‐spectrum immunopathological disease, exacerbated by inflammatory co‐morbidities. A better understanding of mechanisms underpinning virus‐associated inflammation is required to develop effective therapeutics. Here, we discover that SARS‐CoV‐2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG‐I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS‐CoV‐2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS‐CoV‐2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation‐associated COVID‐19.     

A functional assay for serum detection of antibodies against SARS‐CoV‐2 nucleoprotein

The humoral immune response to SARS‐CoV‐2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N‐antibody activity. Here, we present a simple in vitro method called EDNA (electroporated‐antibody‐dependent neutralization assay) that provides a quantitative measure of N‐antibody activity in unpurified serum from SARS‐CoV‐2 convalescents. We show that N antibodies neutralize SARS‐CoV‐2 intracellularly and cell‐autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N‐antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N‐antibody and N‐specific T‐cell activity correlates within individuals, suggesting N antibodies may protect against SARS‐CoV‐2 by promoting antigen presentation. This work highlights the potential benefits of N‐based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.     

Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

Malformations of human cortical development (MCD) can cause severe disabilities. The lack of human‐specific models hampers our understanding of the molecular underpinnings of the intricate processes leading to MCD. Here, we use cerebral organoids derived from patients and genome edited‐induced pluripotent stem cells to address pathophysiological changes associated with a complex MCD caused by mutations in the echinoderm microtubule‐associated protein‐like 1 (EML1) gene. EML1‐deficient organoids display ectopic neural rosettes at the basal side of the ventricular zone areas and clusters of heterotopic neurons. Single‐cell RNA sequencing shows an upregulation of basal radial glial (RG) markers and human‐specific extracellular matrix components in the ectopic cell population. Gene ontology and molecular analyses suggest that ectopic progenitor cells originate from perturbed apical RG cell behavior and yes‐associated protein 1 (YAP1)‐triggered expansion. Our data highlight a progenitor origin of EML1 mutation‐induced MCD and provide new mechanistic insight into the human disease pathology.     

Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS‐CoV‐2. Notably, neutralizing antibodies against SARS‐CoV‐2 isolated from COVID‐19 patients interfered with SARS‐CoV‐2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS‐CoV‐2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS‐CoV‐2, both DC subsets efficiently captured SARS‐CoV‐2 via heparan sulfate proteoglycans and transmitted the virus to ACE2‐positive cells. Notably, human primary nasal cells were infected by SARS‐CoV‐2, and infection was blocked by pre‐treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS‐CoV‐2 infection.     

Construction of miRNA‐lncRNA‐mRNA co‐expression network affecting EMT‐mediated cisplatin resistance in ovarian cancer

Platinum resistance is one of the major concerns in ovarian cancer treatment. Recent evidence shows the critical role of epithelial–mesenchymal transition (EMT) in this resistance. Epithelial‐like ovarian cancer cells show decreased sensitivity to cisplatin after cisplatin treatment. Our study prospected the association between epithelial phenotype and response to cisplatin in ovarian cancer. Microarray dataset {"type":"entrez-geo","attrs":{"text":"GSE47856","term_id":"47856"}}GSE47856 was acquired from the GEO database. After identifying differentially expressed genes (DEGs) between epithelial‐like and mesenchymal‐like cells, the module identification analysis was performed using weighted gene co‐expression network analysis (WGCNA). The gene ontology (GO) and pathway analyses of the most considerable modules were performed. The protein–protein interaction network was also constructed. The hub genes were specified using Cytoscape plugins MCODE and cytoHubba, followed by the survival analysis and data validation. Finally, the co‐expression of miRNA‐lncRNA‐TF with the hub genes was reconstructed. The co‐expression network analysis suggests 20 modules relating to the Epithelial phenotype. The antiquewhite4, brown and darkmagenta modules are the most significant non‐preserved modules in the Epithelial phenotype and contain the most differentially expressed genes. GO, and KEGG pathway enrichment analyses on these modules divulge that these genes were primarily enriched in the focal adhesion, DNA replication pathways and stress response processes. ROC curve and overall survival rate analysis show that the co‐expression pattern of the brown module''s hub genes could be a potential prognostic biomarker for ovarian cancer cisplatin resistance.     

Compartment‐specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell‐specific productivity for IgG1 producing CHO cells after MTA treatment

Increasing cell‐specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5′‐deoxy‐5′‐(methylthio)adenosine (MTA), a chemical degradation product of S‐(5′‐adenosyl)‐ʟ‐methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1‐producing CHO cells by 50%. Compartment‐specific ¹³C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell‐cycle arrest and increased cell volumes. Carbon fluxes into the pentose‐phosphate pathway increased 22 fold in MTA‐treated cells compared to that in non‐MTA‐treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α‐ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA‐treated cells improved three fold compared to that in non‐MTA‐treated cells, which can be regarded as a major factor for explaining the boosted CSPs.     

Hsa_circ_0003945 promotes progression of hepatocellular carcinoma by mediating miR‐34c‐5p/LGR4/β‐catenin axis activity

Accumulating evidence suggests that circular RNAs (circRNAs) play essential roles in regulating cancer progression, but many circRNAs in hepatocellular carcinoma (HCC) remain unknown. Dysregulated circRNAs in HCC were identified through bioinformatics analysis of Gene Expression Omnibus data sets. Quantitative real‐time PCR (qRT‐PCR), Sanger sequencing, RNase R digestion and actinomycin D treatment were conducted to confirm the characterization of circRNAs. CCK‐8, wound‐healing and Transwell assays were performed to assess the functional roles of Hsa_circ_0003945 (Circ_0003945) in HCC cell lines. Subcellular fractionation and fluorescence in situ hybridization (FISH) were performed to locate Circ_0003945 in HCC cells. Dual‐luciferase reporter assay was executed to verify the binding of Circ_0003945 to microRNAs (miRNAs) or the miRNAs to their target genes. In this study, we found that Circ_0003945 was upregulated in HCC tissue, and higher Circ_0003945 expression was positively correlated with tumour size and tumour stage. Furthermore, high plasma levels of circulating Circ_0003945 were confirmed in HCC patients compared with those in non‐HCC groups. The functional experiments revealed that overexpression or knockdown of Circ_0003945 promoted or attenuated tumour growth and migration, respectively. Mechanistically, Circ_0003945 might exert as a miR‐34c‐5p sponge to upregulate the expression of leucine‐rich repeat‐containing G protein‐coupled receptor 4 (LGR4), activating the β‐catenin pathway, and finally facilitating HCC progression. Additionally, a β‐catenin activator could reverse the effect of Circ_0003945 knockdown. In conclusion, Circ_0003945 exerts a tumour‐promoting role in HCC cells by regulating the miR‐34c‐5p/LGR4/β‐catenin axis, which may be a potential target for HCC therapy.     

Molecular and structural mechanisms of ZZ domain‐mediated cargo selection by Nbr1

In selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast Schizosaccharomyces pombe, a selective autophagy pathway termed Nbr1‐mediated vacuolar targeting (NVT) employs Nbr1, an autophagy receptor conserved across eukaryotes including humans, to target cytosolic hydrolases into the vacuole. Here, we identify two new NVT cargos, the mannosidase Ams1 and the aminopeptidase Ape4, that bind competitively to the first ZZ domain of Nbr1 (Nbr1‐ZZ1). High‐resolution cryo‐EM analyses reveal how a single ZZ domain recognizes two distinct protein cargos. Nbr1‐ZZ1 not only recognizes the N‐termini of cargos via a conserved acidic pocket, similar to other characterized ZZ domains, but also engages additional parts of cargos in a cargo‐specific manner. Our findings unveil a single‐domain bispecific mechanism of autophagy cargo recognition, elucidate its underlying structural basis, and expand the understanding of ZZ domain‐mediated protein–protein interactions.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

CFTR interactome mapping using the mammalian membrane two‐hybrid high‐throughput screening system

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high‐throughput screening variant of the Mammalian Membrane Two‐Hybrid (MaMTH‐HTS) to map the protein–protein interactions of wild‐type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient‐specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient‐derived intestinal organoids has a significant effect on CFTR functional expression.