摘要：Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate inEscherichia coliowing to the size and occasional instability of the genome1,2,3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of theCoronaviridae,FlaviviridaeandPneumoviridaefamilies. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step inSaccharomyces cerevisiaeusing transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

摘要：In precision medicine, extracellular vesicles (EVs) are promising intracellular drug delivery vehicles. The development of a quantitative analysis approach will provide valuable information from the perspective of cell biology and system design for drug delivery. Previous studies have reported quantitative methods to analyze the relative uptake or fusion of EVs to recipient cells. However, relatively few methods have enabled the simultaneous evaluation of the "number" of EVs taken up by recipient cells and those that fuse with cellular membranes. In this study, we report a simple quantitative method based on the NanoBiT system to quantify the uptake and fusion of small and large EVs (sEVs and lEVs, respectively). We assessed the abundance of these two subtypes of EVs and determined that lEVs may be more effective vehicles for transporting cargo to recipient cells. The results also indicated that both sEVs and lEVs have very low fusogenic activity, which can be improved in the presence of a fusogenic protein.

摘要：Proteolysis-targeting chimeras (PROTACs) bring a protein of interest (POI) into spatial proximity of an E3 ubiquitinligase, promoting POIubiquitylationand proteasomal degradation. PROTACs rely on endogenous cellular machinery to mediate POI degradation, therefore the subcellular location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether the subcellular context of the POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12F36V(dTAG) constructs consisting of varying localization signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- orcereblon(CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localized to the nucleus, cytoplasm,outer mitochondrial membrane, endoplasmic reticulum, Golgi,peroxisomeorlysosome. Differentially localized Halo or FKBP12F36Vproteins displayed varying levels of degradation using the same respective PROTACs, suggesting therefore that the subcellular context of the POI can influence the efficacy of PROTAC-mediated POI degradation.

摘要：Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

摘要：The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.

摘要：Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton's Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.

摘要：The advent of immune checkpoint inhibition (ICI) using antibodies against PD1 and its ligand PDL1 has prompted substantial efforts to develop complementary drugs. Although many of these are antibodies directed against additional checkpoint proteins, there is an increasing interest in small-molecule immuno-oncology drugs that address intracellular pathways, some of which have recently entered clinical trials. In parallel, small molecules that target pro-tumorigenic pathways in cancer cells and the tumour microenvironment have been found to have immunostimulatory effects that synergize with the action of ICI antibodies, leading to the approval of an increasing number of regimens that combine such drugs. Combinations with small molecules targeting cancermetabolism, cytokine/chemokine and innate immune pathways, and T cell checkpoints are now under investigation. This Review discusses the recent milestones and hurdles encountered in this area of drug development, as well as our views on the best path forward.

摘要：WRKY transcription factors are known to participate in the defence responses of higher plants. However, little is known about the roles of such proteins, especially regarding their functions in the resistance of oilseed rape (Brassica napus) to Sclerotinia sclerotiorum, a necrotrophic fungal pathogen that causes stem rot. In this study, we identified BnWRKY33 as a S. sclerotiorum-responsive gene that positively regulates resistance to this pathogen by enhancing the expression of genes involved in camalexin synthesis and genes regulated by salicylic acid (SA) and jasmonic acid (JA). We also identified a S. sclerotiorum-responsive region in the promoter of BnWRKY33, which we revealed to be a relatively conserved W-box region in the promoters of homologous genes in different species. Using this S. sclerotiorum-responsive region as bait in a yeast one-hybrid assay, we identified another WRKY transcription factor, BnWRKY15, and observed that both BnWRKY15 and BnWRKY33 could bind to this region. In addition, BnWRKY15 overexpression simultaneously increased the susceptibility of B. napus to S. sclerotiorum and down-regulated BnWRKY33 after different durations of infection. Furthermore, BnWRKY15, which contains a transcriptional repression domain, exhibited reduced transactivation ability and could reduce the transactivation ability of BnWRKY33 in Arabidopsis protoplast assays. Therefore, we suggest that the increased susceptibility of BnWRKY15-overexpressing plants results from reduced BnWRKY33 expression, which is due to the inhibition of BnWRKY33 transcriptional activation by BnWRKY15.

摘要：Lipid metabolism is essential for stemness maintenance, self-renewal, and differentiation of stem cells, however, the regulatory function of cholesterol metabolism in erythroid differentiation is poorly studied. In the present study, a critical role for cholesterol homeostasis in terminal erythropoiesis is uncovered. The master transcriptional factor GATA1 binds to Sterol-regulatory element binding protein 2 (SREBP2) to downregulate cholesterol biosynthesis, leading to a gradual reduction in intracellular cholesterol levels. It is further shown that reduced cholesterol functions to block erythroid proliferation via the cholesterol/mTORC1/ribosome biogenesis axis, which coordinates cell cycle exit in the late stages of erythroid differentiation. The interaction of GATA1 and SREBP2 also provides a feedback loop for regulating globin expression through the transcriptional control of NFE2 by SREBP2. Importantly, it is shown that disrupting intracellular cholesterol hemostasis resulted in defect of terminal erythroid differentiation in vivo. These findings demonstrate that fine-tuning of cholesterol homeostasis emerges as a key mechanism for regulating erythropoiesis.

摘要：Smad nuclear-interacting protein 1 (SNIP1) is a transcription repressor related to the TGF-β signaling pathway and associates with c-MYC, a key regulator of cell proliferation and tumor development. Currently, the mechanism by which SNIP1 regulates tumorigenesis and cancer metastasis is unknown. Here, we identify that SNIP1 is a non-histone substrate of lysine methyltransferase KMT5A, which undergoes KMT5A-mediated mono-methylation to promote breast cancer cell growth, invasion and lung metastasis. Mechanistically, we show KMT5A-mediated K301 methylation of SNIP1 represents a sensing signal to release histone acetyltransferase KAT2A and promotes the interaction of c-MYC and KAT2A, and the recruitment of c-MYC/KAT2A complex to promoter of c-MYC targets. This event ultimately inhibits the Hippo kinase cascade to enhance triple-negative breast cancer (TNBC) metastasis by transcriptionally activating MARK4. Co-inhibition of KMT5A catalytic activity and YAP in TNBC xenograft-bearing animals attenuates breast cancer metastasis and increases survival. Collectively, this study presents an KMT5A methylation-dependent regulatory mechanism governing oncogenic function of SNIP1.