In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, ...reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. We have used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly we observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, we found a major role for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome.
Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion ...injury. The level of lipocalin‐2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose‐dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke‐reperfusion injury.
Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling ...intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1’s endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra−/− background. Conversely, IL-17-dependent pathology in Zc3h12a+/− mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3′ UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.
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•MCPIP1 (Regnase-1) is a feedback inhibitor of IL-17 signal transduction•MCPIP1 deficiency enhances immunity to fungi but exacerbates pathology in EAE•MCPIP1 impairs activation of certain IL-17 target promoters by degrading IκBζ mRNA•MCPIP1 degrades transcripts encoding IL-17R subunits independently of the 3′ UTR
Interleukin-17 (IL-17) induces pathology in autoimmunity and infections and therefore its activity must be tightly regulated. Gaffen and colleagues report that the endoribonuclease MCPIP1 degrades IL-17-induced transcripts, including Il6 and Nfkbiz (Iκβζ), to negatively regulate IL-17 receptor signaling both in fungal immunity and experimental autoimmune encephalomyelitis.
Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore ...enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.
High-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the ...bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved.
Human umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed.
The addition of HDL
or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL
had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex.
Altogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.
Objective
Sepsis is the most common trigger for acute kidney injury (AKI) in critically ill patients. We sought to determine whether there are unique patterns to plasma and urine neutrophil ...gelatinase-associated lipocalin (NGAL) in septic compared with non-septic AKI.
Design
Prospective observational study.
Setting
Two adult ICUs in Melbourne, Australia.
Patients
Critically ill patients with septic and non-septic AKI.
Interventions
None.
Measurements and main results
Blood and urine specimens collected at enrollment, 12, 24 and 48 h to measure plasma and urine NGAL. Eighty-three patients were enrolled (septic
n
= 43). Septic AKI patients had more co-morbid disease (
p
= 0.005), emergency surgical admissions (
p
< 0.001), higher illness severity (
p
= 0.008), more organ dysfunction (
p
= 0.008) and higher white blood cell counts (
p
= 0.01). There were no differences at enrollment between groups in AKI severity. Septic AKI was associated with significantly higher plasma (293 vs. 166 ng/ml) and urine (204 vs. 39 ng/mg creatinine) NGAL at enrollment compared with non-septic AKI (
p
< 0.001). Urine NGAL remained higher in septic compared with non-septic AKI at 12 h (
p
< 0.001) and 24 h (
p
< 0.001). Plasma NGAL showed fair discrimination for AKI progression (area under receiver-operator characteristic curve 0.71) and renal replacement therapy (AuROC 0.78). Although urine NGAL performed less well (AuROC 0.70, 0.70), peak urine NGAL predicted AKI progression better in non-septic AKI (AuROC 0.82).
Conclusion
Septic AKI patients have higher detectable plasma and urine NGAL compared with non-septic AKI patients. These differences in NGAL values in septic AKI may have diagnostic and clinical relevance as well as pathogenetic implications.
The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor ...diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6Å resolution and reveals binding to the gfcc′ sheet and cc′ loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved β-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.
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► The ED-B of Fn is relevant for tumor diagnosis and therapy. ► Anticalins were selected from a novel random library based on human Lcn2. ► These Anticalins specifically recognize the ED-B in the context of its flanking domains. ► The stably monomeric Anticalins have high affinities and stain ED-B-positive cells. ► X-ray analysis of the Anticalin N7A bound to Fn7B8 reveals new structural insights.
The lipocalin proteins (lipocalins) are a large family of small proteins characterized by low sequence similarity and highly conserved crystal structures. Lipocalins have been found to play important ...roles in many human diseases. For this reason, a systemic analysis of the molecular properties of human lipocalins is essential. In this study, human lipocalins were found to contain four structurally conserved regions (SCRs) and could be divided into two subgroups. A human lipocalin protein-protein interaction network (PPIN) was constructed and integrated with their expression data in esophageal carcinoma. Many lipocalins showed obvious co-expression patterns in esophageal carcinoma. Their subcellular distributions also suggested these lipocalins may transfer signals from the extracellular space to the nucleus using the pathway-like paths. These analyses also expanded our knowledge about this human ancient protein family in the background of esophageal carcinoma.
Abstract Background Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by an irreversible cognitive decline and neuronal loss associated with neurofibrillary tangles ...and senile plaques. Mild cognitive impairment (MCI) is a prodromal stage of AD and is associated with memory loss and a high risk of developing AD. Lipocalin 2 (LCN2) is an acute phase protein. Our previous studies have shown that exposure to inflammatory stimuli resulted in elevated LCN2 levels in brain microglia and astrocytes implicating LCN2 in brain inflammation. Therefore, we hypothesize that there may be a significant change in the plasma LCN2 levels in patients with MCI and AD when compared to healthy control subjects. Methods Forty-one patients with MCI, 62 patients with AD and 38 healthy elderly control subjects were recruited for this study. They were given a comprehensive battery of neuropsychological tests including a mini-mental status examination (MMSE) and clinical dementia rating (CDR). A variety of clinical information was collected from the semi-structured questionnaire administered. The LCN2 levels were measured using a specific enzyme-linked immunosorbent assay in the plasma, which had been collected early in the morning after overnight fasting. Results The LCN2 levels were significantly higher in MCI patients compared to the healthy control subjects and AD patients control vs. MCI (p = 0.005); MCI vs. AD (p = 0.009). There was a significant negative correlation between the LCN2 levels and CDR scores (r = − 0.245, p = 0.014), and there was a positive correlation between the LCN2 levels and MMSE scores (r = 0.317, p = 0.001) among all of the MCI and AD patients. Conclusion MCI represents a prodromal stage of AD, and inflammation occurs as one of the earliest pathological events in AD. Thus, increased plasma LCN2 levels during MCI could be helpful in predicting the progression from MCI to AD.
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