LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis

Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.     

Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes

Autophagy is a major intracellular trafficking pathway that delivers proteins and organelles from the cytoplasm into lysosomes for consequential degradation and recycling. Mammalian Atg8s are key autophagic factors that undergo a unique ubiquitin-like conjugation to the lipid phase of the autophagosomal membrane. In addition to their activity in autophagosome formation, several Atg8s directly bind p62/SQSTM1. Here we show that LC3 and GATE-16 differ in their mode of p62 binding. While the soluble form of both LC3 and GATE-16 bind p62, only the lipidated form of LC3 is directly involved in p62 recruitment into autophagosomes. Moreover, by utilizing chimeras of LC3 and GATE-16 where their N-terminus was swapped, we determined the regions responsible for this differential binding. Accordingly, we found that the chimera of GATE-16 containing the LC3 N-terminal region acts similarly to wild-type LC3 in recruiting p62 into autophagosomes. We therefore propose that LC3 is responsible for the final stages of p62 incorporation into autophagosomes, a process selectively mediated by its N-terminus.     

Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis

Background

The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity.

Methodology/Principal Findings

Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1^−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens.

Conclusions

Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.     

An essential role for diet in exercise-mediated protection against dyslipidemia, inflammation and atherosclerosis in ApoE⁻/⁻ mice

Background

Diet and exercise promote cardiovascular health but their relative contributions to atherosclerosis are not fully known. The transition from a sedentary to active lifestyle requires increased caloric intake to achieve energy balance. Using atherosclerosis-prone ApoE-null mice we sought to determine whether the benefits of exercise for arterial disease are dependent on the food source of the additional calories.

Methods and Results

Mice were fed a high-fat diet (HF) for 4.5 months to initiate atherosclerosis after which time half were continued on HF while the other half were switched to a high protein/fish oil diet (HP). Half of each group underwent voluntary running. Food intake, running distance, body weight, lipids, inflammation markers, and atherosclerotic plaque were quantified. Two-way ANOVA tests were used to assess differences and interactions between groups. Exercised mice ran approximately 6-km per day with no difference between groups. Both groups increased food intake during exercise and there was a significant main effect of exercise F((1,44) = 9.86, p<0.01) without interaction. Diet or exercise produced significant independent effects on body weight (diet: F(1,52) = 6.85, p = 0.012; exercise: F(1,52) = 9.52, p<0.01) with no significant interaction. The combination of HP diet and exercise produced a greater decrease in total cholesterol (F(1, 46) = 7.9, p<0.01) and LDL (F(1, 46) = 7.33, p<0.01) with a large effect on the size of the interaction. HP diet and exercise independently reduced TGL and VLDL (p<0.05 and 0.001 respectively). Interleukin 6 and C-reactive protein were highest in the HF-sedentary group and were significantly reduced by exercise only in this group. Plaque accumulation in the aortic arch, a marker of cardiovascular events was reduced by the HP diet and the effect was significantly potentiated by exercise only in this group resulting in significant plaque regression (F1, 49 = 4.77, p<0.05).

Conclusion

In this model exercise is beneficial to combat dyslipidemia and protect from atherosclerosis only when combined with diet.     

Protein profiling underscores immunological functions of uterine cervical mucus plug in human pregnancy

The cervical mucus plug (CMP) differs from the cervical secretions of non-pregnant women, and is the ultimate sealant of the uterine cavity during pregnancy. Although several studies have analyzed biochemical properties of large glycoproteins in the CMP, comprehensive information about its protein composition is yet unavailable. We hypothesized that protein profiling of the CMP could provide key clues to its physiological functions in pregnancy. For this purpose, five CMPs obtained from women in labor at term were analyzed by LC-MS/MS. Out of 291 total proteins identified, 137 were detected in two or more samples, which included S100A8, S100A9, and complement proteins (C3, C4a, C4b, C6, and C8g). Several proteins, which have not been described in the cervical mucus of non-pregnant women or in cervicovaginal fluids, such as CD81 antigen and pregnancy zone protein, were also identified. Gene ontology analysis of identified proteins showed significant enrichment of 28 biological processes such as 'activation of plasma proteins involved in acute inflammatory response' and 'positive regulation of cholesterol esterification'. We report the proteome of CMPs from pregnant women at term for the first time, and the overall findings strongly suggest an important role for the CMP in the maintenance of pregnancy and parturition.     

Variance in centrality within rock hyrax social networks predicts adult longevity

Background

In communal mammals the levels of social interaction among group members vary considerably. In recent years, biologists have realized that within-group interactions may affect survival of the group members. Several recent studies have demonstrated that the social integration of adult females is positively associated with infant survival, and female longevity is affected by the strength and stability of the individual social bonds. Our aim was to determine the social factors that influence adult longevity in social mammals.

Methodology/Principal Findings

As a model system, we studied the social rock hyrax (Procavia capensis), a plural breeder with low reproductive skew, whose groups are mainly composed of females. We applied network theory using 11 years of behavioral data to quantify the centrality of individuals within groups, and found adult longevity to be inversely correlated to the variance in centrality. In other words, animals in groups with more equal associations lived longer. Individual centrality was not correlated with longevity, implying that social tension may affect all group members and not only the weakest or less connected ones.

Conclusions/Significance

Our novel findings support previous studies emphasizing the adaptive value of social associations and the consequences of inequality among adults within social groups. However, contrary to previous studies, we suggest that it is not the number or strength of associations that an adult individual has (i.e. centrality) that is important, but the overall configuration of social relationships within the group (i.e. centrality SD) that is a key factor in influencing longevity.     

Maturation of escape circuit function during the early adulthood of cockroaches Periplaneta americana

During postembryonic development of insects, sensorimotor pathways, which generate specific behaviors, undergo maturational changes. It is less clear whether such pathways are typically stable, or undergo further maturation, during the adult stage. In the present study, we have examined this issue by multilevel analysis of a simple model system, the escape behavior of the cockroach, from identified synapses to behavior. We show that the escape system is highly responsive immediately after the molt to adulthood, but that the latency of escape responses was not at its typical value immediately after the molt to adult. The latency of escape behavior increased over the first 30 days of adult life, perhaps indicating maturational adjustments of the escape sensorimotor pathway. The first station in the escape circuitry is the synaptic connections between the cercal wind receptors and the giant interneurons. We measured unitary excitatory synaptic potentials between single sensory neurons and an identified giant interneuron (GI(2)). We found a decrease in the synaptic strength between identified cercal hairs from a single column and GI(2) over the first month after the adult molt. Consequently, the latency and the number of action potentials of GI(2) in response to natural stimuli increased and decreased respectively during this time. Thus, we show that both behavioral performance and the wind sensitivity of GI(2) decreased over the first month after molt. We conclude that the cockroach escape system undergoes further sensorimotor maturation over a period of 1 month, and that cellular changes correlate with, or predict, some changes in behavioral performance.