Hepatitis C Virus Induces E6AP-Dependent Degradation of the Retinoblastoma Protein

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer

Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta

Dermal immature adipocytes fightagainstStaphylococcusaureusinfectionby secreting antimicrobial peptidesduring adipogenesis. Zhang et al.demonstrate that activation of the TGF-bpathway suppresses the adipogenicpotential of dermal fibroblasts andtherefore leads to an age-dependent lossof antimicrobial protection fromdermal fat. 皮下脂肪细胞作为皮肤的最后一道防线，有很重要的免疫抗菌功能，分化中脂肪细胞释放大量抗菌肽，这是一种人自源抗生素，能有效地抑制细菌生长。但这一重要的宿主天然免疫功能在发育和老化过程中如何被调控还不为人知。研究人员发现，老化过程中皮肤脂肪丢失是和真皮成纤维细胞（dermal fibroblasts）失去脂肪分化能力密切相关的。真皮成纤维细胞是皮肤深处的特化细胞，可以产生结缔组织并帮助皮肤从损伤中恢复。【Abstract】Dermal fibroblasts (dFBs) resist infection by locallydifferentiating into adipocytes and producing cathe-licidin antimicrobial peptide in response toStaphylo-coccusaureus(S.aureus). Here, we show thatneonatal skin was enriched with adipogenic dFBsand immature dermal fat that highly expressed cath-elicidin. The pool of adipogenic and antimicrobialdFBs declined after birth, leading to an age-depen-dent loss of dermal fat and a decrease in adipogene-sis and cathelidicin production in response to infec-tion. Transforming growth factor beta (TGF-b),which acted on uncommitted embryonic and adultdFBs and inhibited their adipogenic and antimicro-bial function, was identified as a key upstream regu-lator of this process. Furthermore, inhibition of theTGF-breceptor restored the adipogenic and antimi-crobial function of dFBs in culture and increasedresistance of adult mice toS.aureusinfection. Theseresults provide insight into changes that occur in theskin innate immune system between the perinataland adult periods of life.This work was supported by NIH grant R01-AR069653 to L.Z. and R.L.G. and NIH grants R01-AI083358, R01-AR052728, and U19-AI117673 to R.L.G. M.V.P. is supported by a Pew Charitable Trust grant, NIH grants U01-AR073159 and R01-AR067273, National Science Foundation (NSF) grant DMS1763272, and Simons Foundation grant 594598 (to Qing Nie). C.F.G.J. is supported by NSF-GRFP grant DGE-1321846 and MBRS-IMSD training grant GM055246. Y.Z. is supported by NIH grant R01-AI107027. 该项目研究得到了厦门大学双一流启动基金的支持

Disruption of TLR3 Signaling Due to Cleavage of TRIF by the Hepatitis A Virus Protease-Polymerase Processing Intermediate, 3CD

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Ferroptosis Regulated by Hypoxia in Cells

Ferroptosis is an oxidative damage-related, iron-dependent regulated cell death with intracellular lipid peroxide accumulation, which is associated with many physiological and pathological processes. It exhibits unique features that are morphologically, biochemically, and immunologically distinct from other regulated cell death forms. Ferroptosis is regulated by iron metabolism, lipid metabolism, anti-oxidant defense systems, as well as various signal pathways. Hypoxia, which is found in a group of physiological and pathological conditions, can affect multiple cellular functions by activation of the hypoxia-inducible factor (HIF) signaling and other mechanisms. Emerging evidence demonstrated that hypoxia regulates ferroptosis in certain cell types and conditions. In this review, we summarize the basic mechanisms and regulations of ferroptosis and hypoxia, as well as the regulation of ferroptosis by hypoxia in physiological and pathological conditions, which may contribute to the numerous diseases therapies

Study on the Spatial and Temporal Evolution Patterns of Green Innovation Efficiency and Driving Factors in Three Major Urban Agglomerations in China—Based on the Perspective of Economic Geography

Sustainable development has become a global consensus, and green innovation is the key starting point, and it has become a ballast stone and stabilizer for regional ecological environmental protection and high-quality economic development. Based on GIS tools and multi-method models, this paper studies the spatio-temporal characteristics and influence mechanism of green innovation in three major urban agglomerations in China from 2010 to 2019 from the perspective of economic geography. The study found that: (1) the green innovation efficiency of the three major urban agglomerations in China is in a fluctuating upward trend as a whole, with obvious spatial disequilibrium; (2) from the spatial point of view, the characteristics of global spatial agglomeration distribution have positive spatial correlation, and the law of local spatial autocorrelation is obvious, and the spatio-temporal pattern transitions from “low efficiency, big difference” to “high efficiency, small difference” during the study period; (3) there are obvious spatial spillover and diffusion effects on the green innovation efficiency of the three major urban agglomerations in China as a whole. However, the spatial dependence of green innovation efficiency is inconsistent in China’s three major urban agglomerations; the YRD Urban region and the PRD Urban region show a positive impact, while the JJJ Urban region shows a negative impact; (4) the level of economic development, the operating environment of science and technology, and the guiding factors of government system function with significant differences and regional spatial heterogeneity on the efficiency of green innovation in the three major urban agglomerations in China

Aggregation, Fusion, and Leakage of Liposomes Induced by Peptides

Biological membranes are heterogeneous systems. Their functions are closely related to the lipid lateral segregation in the presence of membrane proteins. In this work, we designed two peptides, amphiphilic cationic peptides K₃L₈K₃ and nonamphiphilic peptides K₂₀, and studied their interactions with binary liposomes in different phases (<i>L</i>_α, <i>L</i>_β′, and <i>L</i>_α/<i>L</i>_β′). As mimics of membrane proteins, both K₃L₈K₃ and K₂₀ can cause the liposomes to aggregate, fuse, or leak. These processes were closely related to the phases of liposomes. For the liposomes in <i>L</i>_α phase, heavy aggregation, fusion, and leakage were observed in the presence of either K₂₀ or K₃L₈K₃. For the liposomes in <i>L</i>_β′ phase, neither K₃L₈K₃ nor K₂₀ can induce fusion or leakage. For the liposomes in <i>L</i>_α/<i>L</i>_β′ phase, K₃L₈K₃ caused the liposomes to aggregate, fuse, and leak, while K₂₀ only led to aggregation. The kinetics of aggregation, fusion, and leakage in each phase were recorded, and they were related to the lipid demixing in the presence of the peptide. Our work not only gained insight into the effect of the lipid demixing on the interactions between peptide and membrane, but also helped in developing drug delivery vehicles with liposomes as the platform