The SOCS Box Encodes a Hierarchy of Affinities for Cullin5: Implications for Ubiquitin Ligase Formation and Cytokine Signalling Suppression

The SOCS (suppressors of cytokine signalling) family of proteins inhibits the cytokine-induced signalling cascade in part by promoting the ubiquitination of signalling intermediates that are then targeted for proteasomal degradation. This activity relies upon an interaction between the SOCS box domain, the adapter complex elonginBC and a member of the Cullin family, the scaffold protein of an E3 ubiquitin ligase. In this study, we dissected this interaction in vitro using purified components. We found that all eight SOCS proteins bound Cullin5 but required prior recruitment of elonginBC. Neither SOCS nor elonginBC bound Cullin5 when in isolation. Interestingly, the affinity of each SOCS-elonginBC complex for Cullin5 varied by 2 orders of magnitude across the SOCS family. Unexpectedly, the most potent suppressors of signalling, SOCS-1 and SOCS-3, bound most weakly to the E3 ligase scaffold, with affinities 100- and 10-fold lower, respectively, than the rest of the family. The remaining six SOCS proteins all bound Cullin5 with high affinity (K_d of ∼ 10 nM) due to a slower off-rate and hence a longer half-life of the complex. This difference in affinity may reflect a difference in mode of action as only SOCS-1 and SOCS-3 have been shown to suppress signalling using both SOCS box-dependent and SOCS box-independent mechanisms. This is not the case with the other six SOCS proteins, and our data imply the existence of two distinct subclasses of SOCS proteins with a high affinity for Cullin5, the E3 ligase scaffold, possibly reflecting complete dependence upon ubiquitination for suppression of cytokine signalling.     

Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97

Auto-proteolysis at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is a hallmark of adhesion-GPCRs. Although defects in GPS auto-proteolysis have been linked to genetic disorders, information on its regulation remains elusive. Here, we investigated the GPS proteolysis of CD97, a human leukocyte-restricted and tumor-associated adhesion-GPCR. We found that CD97 is incompletely processed, unlike its close homolog, epidermal growth factor-like module-containing mucin-like hormone receptor 2. A unique pattern of N-glycosylation within the GPS motif of related adhesion-GPCRs was identified. The use of N-glycosylation inhibitors and mutants confirm site-specific N-glycosylation is an important determinant of GPS proteolysis in CD97. Our results suggest that N-glycosylation may regulate the processing of adhesion-GPCRs leading to the production of either cleaved or uncleaved molecules.     

Juvenile hormone and the ontogeny of cockroach aggression

Our previous study [Kou et al., 2008. Juvenile hormone levels are increased in winners of cockroach fights. Horm. Behav. 52, 252–260] showed that the basic principle of the challenge hypothesis (hormone levels can respond to social stimuli to modulate aggression in vertebrates] could be applied to juvenile hormone (JH) levels and aggression in the lobster cockroach Nauphoeta cinerea. In that study, 80- to 85-day-old socially naïve males were used, as fighting is much more easily initiated in these older animals than in younger males, and JH III levels in the dominant were found to be significantly increased after an encounter compared to before the encounter and were significantly higher than those in the subordinates. In N. cinerea, newly emerged males usually show no aggressiveness towards each other and aggression is only initiated after several days of close contact. To investigate the development of aggression from an early age, in the present study, newly emerged males were paired to investigate the relationship between JH levels and aggression. The results showed that injection of JH III significantly increased the probability of the young males being fight winners. In each age group in which aggression was initiated, the dominants had significantly higher JH levels than either the subordinates or the same aged non-fighters. JH injection of subordinates on the day of rank establishment had no effect on the probability of rank switch. These results indicate that, (i) in newly emerged male pairs, JH plays a decisive role in rank establishment and the fact that dominant status is significantly associated with a higher JH titer and subordinate status with a lower JH titer is consistent with the basic principle of the challenge hypothesis, and (ii) after rank establishment, the lack of effect of JH treatment on rank change is consistent with the idea of “social inertia” in vertebrates.     

新的生长激素释放激素类似肽Pro-hGHRH(1-44)-Gly-Gly-Cys的重组和比较活性*

目的:为了寻找高活性和长半衰期生的GHRH类似肽。方法:通过使用独特的酸敏感水解位点Asp-Pro的原核表达系统,构建了新的Pro-hGHRH(1-44)-Gly-Gly-Cys类似肽。通过重组细菌裂解、包含体洗涤、乙醇分级沉淀、酸水解、SP-Sephadex C-25和Sephadex G-10柱层析等技术,纯化了高纯度的Pro-hGHRH(1-44)-Gly-Gly-Cys肽。通过使用SDS-PAGE、离子化质谱、雌大鼠垂体和人流产胎儿垂体,测定了多肽的纯度、分子量、生长激素释放活性。结果:Pro-hGHRH(1-44)-Gly-Gly-Cys肽分子量5373Da与实际值吻合,0.1~10 μg/ml的肽剂量不论是对人垂体还是大鼠垂体都增加了垂体生长激素的释放,大鼠垂体生长激素的释放具有剂量依赖性。与标准的hGHRH(1-40)肽比较,新的类似肽有较高的GH释放活性。结果也显示了,Pro-GHRH(1-44)-Gly-Gly-Cys与Pro-hGHRH(1-44)肽的GH释放活性无统计学差异。结论：新的类似肽有较好的生长激素释放活性、功能选择性和种属特异性。     

The growth hormone receptor gene‐disrupted mouse fails to respond to an intermittent fasting diet

The interaction of longevity‐conferring genes with longevity‐conferring diets is poorly understood. The growth hormone receptor gene‐disrupted (GHR‐KO) mouse is long lived; and this longevity is not responsive to 30% caloric restriction, in contrast to wild‐type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction, we subjected GHR‐KO mice to a different dietary restriction regimen, an intermittent fasting diet. The intermittent fasting diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR‐KO mice. From the results of two paradigms of dietary restriction, we postulate that GHR‐KO mice would be resistant to any manner of dietary restriction; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan extending potential of an intervention.     

Wool fibre crimp is determined by mitotic asymmetry and position of final keratinisation and not ortho- and para-cortical cell segmentation

Crimp, a distinguishing feature of sheep fibres, significantly affects wool value, processing and final fabric attributes. Several explanations for fibre bending have been proposed. Most concentrate on relative differences in the physicochemical properties of the cortical cells, which comprise the bulk of the fibre. However, the associations between cortical properties and fibre crimp are not consistent and may not reflect the underlying causation of fibre curvature (FC). We have formulated a mechanistic model in which fibre shape is dictated primarily by the degree of asymmetry in cell supply from the follicle bulb, and the point at which keratinisation is completed within the follicle. If this hypothesis is correct, one would anticipate that most variations in fibre crimp would be accounted for by quantitative differences in both the degree of mitotic asymmetry in follicle bulbs and the distance from the bulb to the point at which keratinisation is completed. To test this hypothesis, we took skin biopsies from Merino sheep from sites producing wool differing widely in fibre crimp frequency and FC. Mitotic asymmetry in follicle bulbs was measured using a DNA-labelling technique and the site of final keratinisation was defined by picric acid staining of the fibre. The proportion of para- to ortho-cortical cell area was determined in the cross-sections of fibres within biopsy samples. Mitotic asymmetry in the follicle bulb accounted for 0.64 (P < 0.0001) of the total variance in objectively measured FC, while the point of final keratinisation of the fibre accounted for an additional 0.05 (P < 0.05) of the variance. There was no association between ortho- to para-cortical cell ratio and FC. FC was positively associated with a subjective follicle curvature score (P < 0.01). We conclude that fibre crimp is caused predominantly by asymmetric cell division in follicles that are highly curved. Differential pressures exerted by the subsequent asymmetric cell supply and cell hardening in the lower follicle cause fibre bending. The extent of bending is then modulated by the point at which keratinisation is completed; later hardening means the fibre remains pliable for longer, thereby reducing the pressure differential and reducing fibre bending. This means that even highly asymmetric follicles may produce a straight fibre if keratinisation is sufficiently delayed, as is the case in deficiencies of zinc and copper, or when keratinisation is perturbed by transgenesis. The model presented here can account for the many variations in fibre shape found in mammals.     

Hypothalamic lipotoxicity and the metabolic syndrome

Ectopic accumulation of lipids in peripheral tissues, such as pancreatic β cells, liver, heart and skeletal muscle, leads to lipotoxicity, a process that contributes substantially to the pathophysiology of insulin resistance, type 2 diabetes, steatotic liver disease and heart failure. Current evidence has demonstrated that hypothalamic sensing of circulating lipids and modulation of hypothalamic endogenous fatty acid and lipid metabolism are two bona fide mechanisms modulating energy homeostasis at the whole body level. Key enzymes, such as AMP-activated protein kinase (AMPK) and fatty acid synthase (FAS), as well as intermediate metabolites, such as malonyl-CoA and long-chain fatty acids-CoA (LCFAs-CoA), play a major role in this neuronal network, integrating peripheral signals with classical neuropeptide-based mechanisms. However, one key question to be addressed is whether impairment of lipid metabolism and accumulation of specific lipid species in the hypothalamus, leading to lipotoxicity, have deleterious effects on hypothalamic neurons. In this review, we summarize what is known about hypothalamic lipid metabolism with focus on the events associated to lipotoxicity, such as endoplasmic reticulum (ER) stress in the hypothalamus. A better understanding of these molecular mechanisms will help to identify new drug targets for the treatment of obesity and metabolic syndrome.     

半胱胺促进空怀奶山羊乳腺发育的试验研究

目的：研究半胱胺对空怀奶山羊乳腺发育的影响。方法：选6只空怀奶山羊进行自身前后对照试验。对照期饲喂基础日粮,试验期饲喂基础日粮加CS。结果：与对照期相比,试验期山羊乳腺组织的DNA含量明显升高,RNA含量明显下降,故RNA／DNA值显著下降。试验期血浆GH和PRL水平分别提高61．6％和31．6％。组织学观察发现,对照期乳腺的腺泡很少发育,几乎看不到乳导管;试验期乳腺有明显导管生长和少量腺泡发育。结论：半胱胺能够在一定程度上促进空怀奶山羊乳腺发育。     

Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing

Regulatory peptides represent a diverse group of messenger molecules. In insects, they are produced by endocrine cells as well as secretory neurones within the CNS. Many regulatory peptides are released as hormones into the haemolymph to regulate, for example, diuresis, heartbeat or ecdysis behaviour. Hormonal release of neuropeptides takes place at specialized organs, so-called neurohaemal organs. We have performed a mass spectrometric characterization of the peptide complement of the main neurohaemal organs and endocrine cells of the Drosophila melanogaster larva to gain insight into the hormonal communication possibilities of the fruit fly. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and MALDI-TOF-TOF tandem mass spectrometry, we detected 23 different peptides of which five were unpredicted by previous genome screenings. We also found a hitherto unknown peptide product of the capa gene in the ring gland and transverse nerves, suggesting that it might be released as hormone. Our results show that the peptidome of the neurohaemal organs is tagma-specific and does not change during metamorphosis. We also provide evidence for the first case of differential prohormone processing in Drosophila.