Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-alpha/beta and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3' non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP-RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.     

Direct observation of the mechanochemical coupling in myosin Va during processive movement

Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.     

Heterochromatin links to centromeric protection by recruiting shugoshin

The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6 cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.     

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.     

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response. Rab5 is known as a key regulator of the early endocytic pathway and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.     

A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T --> C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-beta1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-beta1-mediated induction of cartilage matrix genes through direct interaction with TGF-beta1 and inhibition of TGF-beta1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-beta1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-beta signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-beta.     

Ca2+及cAMP调控的神经递质与激素释放

神经递质和激素的释放受胞内诸多信号通路的调控。这两种内源物质的释放都由依赖于Ca²⁺浓度的快速引发机制介导,相对于其他介导因素,[Ca²⁺]占主导地位。神经递质和激素释放的调控大多是通过调节释放小泡数量实现的。尽管在处于静息状态的神经末梢中,释放小泡的数量相对恒定,但当释放小泡被胞吐作用“消耗”后,其再生速度受调控作用介导而发生改变,从而导致小泡数量“稳中有变”的动态调控过程。这一动态调控作用表明了“消耗”与“再生”之间的平衡机制。     

Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

Background： The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein （LDL） in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. Methods： We have studied the effect of fetal calf serum （FCS）, which we considered a source of LDL, on the adhesion of monocytes to endothelial cells （ECs） by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells （EC monoculture） and a co-culture with bovine aortic smooth muscle cells （EC-SMC co-culture）. Results： It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP- 1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells. Conclusion： The results suggest that the elevation of the LDL （cholesterol） level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.