Journal: Cell discovery
The prokaryotic CRISPR-Cas adaptive immune systems provide valuable resources to develop genome editing tools, such as CRISPR-Cas9 and CRISPR-Cas12a/Cpf1. Recently, CRISPR-Cas12b/C2c1, a distinct type V-B system, has been characterized as a dual-RNA-guided DNA endonuclease system. Though being active in vitro, its cleavage activity at endogenous genome remains to be explored. Furthermore, the optimal cleavage temperature of the reported Cas12b orthologs is higher than 40 °C, which is unsuitable for mammalian applications. Here, we report the identification of a Cas12b system from the Alicyclobacillus acidiphilus (AaCas12b), which maintains optimal nuclease activity over a wide temperature range (31 °C-59 °C). AaCas12b can be repurposed to engineer mammalian genomes for versatile applications, including single and multiplex genome editing, gene activation, and generation of gene mutant mouse models. Moreover, whole-genome sequencing reveals high specificity and minimal off-target effects of AaCas12b-meditated genome editing. Our findings establish CRISPR-Cas12b as a versatile tool for mammalian genome engineering.
5-hydroxytryptamine (5-HT, also known as serotonin) regulates many physiological processes through the 5-HT receptor family. Here we report the crystal structure of 5-HT1B subtype receptor (5-HT1BR) bound to the psychotropic serotonin receptor inverse agonist methiothepin (MT). Crystallization was facilitated by replacing ICL3 with a novel optimized variant of BRIL (OB1) that enhances the formation of intermolecular polar interactions, making OB1 a potential useful tool for structural studies of membrane proteins. Unlike the agonist ergotamine (ERG), MT occupies only the conserved orthosteric binding pocket, explaining the wide spectrum effect of MT on serotonin receptors. Compared with ERG, MT shifts toward TM6 and sterically pushes residues W3276.48, F3306.50 and F3316.51 from inside the orthosteric binding pocket, leading to an outward movement of the extracellular end and a corresponding inward shift of the intracellular end of TM6, a feature shared by other reported inactive G protein-coupled receptor (GPCR) structures. Together with the previous agonist-bound serotonin receptor structures, the inverse agonist-bound 5-HT1BR structure identifies a basis for the ligand-mediated switch of 5-HT1BR activity and provides a structural understanding of the inactivation mechanism of 5-HT1BR and some other class A GPCRs, characterized by ligand-induced outward movement of the extracellular end of TM6 that is coupled with inward movement of the cytoplasmic end of this helix.
Human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV) and 2019 novel coronavirus (2019-nCoV, also known as SARS-CoV-2), lead global epidemics with high morbidity and mortality. However, there are currently no effective drugs targeting 2019-nCoV/SARS-CoV-2. Drug repurposing, representing as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. In this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the HCoV-host interactome and drug targets in the human protein-protein interaction network. Phylogenetic analyses of 15 HCoV whole genomes reveal that 2019-nCoV/SARS-CoV-2 shares the highest nucleotide sequence identity with SARS-CoV (79.7%). Specifically, the envelope and nucleocapsid proteins of 2019-nCoV/SARS-CoV-2 are two evolutionarily conserved regions, having the sequence identities of 96% and 89.6%, respectively, compared to SARS-CoV. Using network proximity analyses of drug targets and HCoV-host interactions in the human interactome, we prioritize 16 potential anti-HCoV repurposable drugs (e.g., melatonin, mercaptopurine, and sirolimus) that are further validated by enrichment analyses of drug-gene signatures and HCoV-induced transcriptomics data in human cell lines. We further identify three potential drug combinations (e.g., sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the “Complementary Exposure” pattern: the targets of the drugs both hit the HCoV-host subnetwork, but target separate neighborhoods in the human interactome network. In summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations targeting 2019-nCoV/SARS-CoV-2.
The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.
An outbreak of clusters of viral pneumonia due to a novel coronavirus (2019-nCoV/SARS-CoV-2) happened in Wuhan, Hubei Province in China in December 2019. Since the outbreak, several groups reported estimated R0 of Coronavirus Disease 2019 (COVID-19) and generated valuable prediction for the early phase of this outbreak. After implementation of strict prevention and control measures in China, new estimation is needed. An infectious disease dynamics SEIR (Susceptible, Exposed, Infectious, and Removed) model was applied to estimate the epidemic trend in Wuhan, China under two assumptions of R t . In the first assumption, R t was assumed to maintain over 1. The estimated number of infections would continue to increase throughout February without any indication of dropping with R t = 1.9, 2.6, or 3.1. The number of infections would reach 11,044, 70,258, and 227,989, respectively, by 29 February 2020. In the second assumption, R t was assumed to gradually decrease at different phases from high level of transmission (R t = 3.1, 2.6, and 1.9) to below 1 (R t = 0.9 or 0.5) owing to increasingly implemented public health intervention. Several phases were divided by the dates when various levels of prevention and control measures were taken in effect in Wuhan. The estimated number of infections would reach the peak in late February, which is 58,077-84,520 or 55,869-81,393. Whether or not the peak of the number of infections would occur in February 2020 may be an important index for evaluating the sufficiency of the current measures taken in China. Regardless of the occurrence of the peak, the currently strict measures in Wuhan should be continuously implemented and necessary strict public health measures should be applied in other locations in China with high number of COVID-19 cases, in order to reduce R t to an ideal level and control the infection.
Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.
The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, while it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have determined the complex structure between Rpn13NTD and K48-diUb. In this structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge-reversal mutation in Rpn13NTD weakens the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, physical blockage of the access of the distal Ub to Rpn13NTD with a proximity-attached Ub monomer can disrupt the interaction between Rpn13 and K48-diUb. Taken together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.
COVID-19, caused by SARS-CoV-2, has recently affected over 1,200,000 people and killed more than 60,000. The key immune cell subsets change and their states during the course of COVID-19 remain unclear. We sought to comprehensively characterize the transcriptional changes in peripheral blood mononuclear cells during the recovery stage of COVID-19 by single-cell RNA sequencing technique. It was found that T cells decreased remarkably, whereas monocytes increased in patients in the early recovery stage (ERS) of COVID-19. There was an increased ratio of classical CD14++ monocytes with high inflammatory gene expression as well as a greater abundance of CD14++IL1β+ monocytes in the ERS. CD4+ T cells and CD8+ T cells decreased significantly and expressed high levels of inflammatory genes in the ERS. Among the B cells, the plasma cells increased remarkably, whereas the naïve B cells decreased. Several novel B cell-receptor (BCR) changes were identified, such as IGHV3-23 and IGHV3-7, and isotypes (IGHV3-15, IGHV3-30, and IGKV3-11) previously used for virus vaccine development were confirmed. The strongest pairing frequencies, IGHV3-23-IGHJ4, indicated a monoclonal state associated with SARS-CoV-2 specificity, which had not been reported yet. Furthermore, integrated analysis predicted that IL-1β and M-CSF may be novel candidate target genes for inflammatory storm and that TNFSF13, IL-18, IL-2, and IL-4 may be beneficial for the recovery of COVID-19 patients. Our study provides the first evidence of an inflammatory immune signature in the ERS, suggesting COVID-19 patients are still vulnerable after hospital discharge. Identification of novel BCR signaling may lead to the development of vaccines and antibodies for the treatment of COVID-19.
Elucidating the origin of microglia is crucial for understanding their functions and homeostasis. Previous study has indicated that Nestin-positive progenitor cells differentiate into microglia and replenish the brain after depleting most brain microglia. Microglia have also shown the capacity to repopulate the retina after eliminating all retinal microglia. However, the origin(s) of repopulated retinal microglia is/are unknown. In this study, we aim to investigate the origins of repopulated microglia in the retina. Interestingly, we find that repopulated retinal microglia are not derived from Nestin-positive progenitor cells. Instead, they have two origins: the center-emerging microglia are derived from residual microglia in the optic nerve and the periphery-emerging microglia are derived from macrophages in the ciliary body/iris. Therefore, we have for the first time identified the extra-retinal origins of microglia in the adult mammalian retina by using a model of microglial repopulation, which may shed light on the target exploration of therapeutic interventions for retinal degenerative disorders.
Although conventional genetic modification approaches for protein knockdown work very successfully due to the increasing use of CRISPR/Cas9, effective techniques for achieving protein depletion in adult animals, especially in large animals such as non-human primates, are lacking. Here, we report a chemical approach based on PROTACs technology that efficiently and quickly knocks down FKBP12 (12-kDa FK506-binding) protein globally in vivo. Both intraperitoneal and oral administration led to rapid, robust, and reversible FKBP12 degradation in mice. The efficiency and practicality of this method were successfully demonstrated in both large and small animals (mice, rats, Bama pigs, and rhesus monkeys). Furthermore, we showed this approach can also be applied to effectively knockdown other target proteins such as Bruton’s tyrosine kinase (BTK). This chemical protein knockdown strategy provides a powerful research tool for gene function studies in animals, particularly in large animals, for which gene-targeted knockout strategies may remain unfeasible.