Folding of an Intrinsically Disordered Iron-Binding Peptide in Response to Sedimentation Revealed by Cryo-EM.

Biomineralization is mediated by specialized proteins that guide and control mineral sedimentation. In many cases, the active regions of these biomineralization proteins are intrinsically disordered. High-resolution structures of these proteins while they interact with minerals are essential for understanding biomineralization processes and the function of intrinsically disordered proteins (IDPs). Here we used the cavity of ferritin as a nanoreactor where the interaction between M6A, an intrinsically disordered iron-binding domain, and an iron oxide particle was visualized at high resolution by cryo-EM. Taking advantage of the differences in the electron-dose sensitivity of the protein and the iron oxide particles, we developed a method to determine the irregular shape of the particles found in our density maps. We found that the folding of M6A correlates with the detection of mineral particles in its vicinity. M6A interacts with the iron oxide particles through its C-terminal side, resulting in the stabilization of a helix at its N-terminal side. The stabilization of the helix at a region that is not in direct contact with the iron oxide particle demonstrates the ability of IDPs to respond to signals from their surroundings by conformational changes. These findings provide the first glimpse toward the long-suspected mechanism for biomineralization protein control over mineral microstructure, where unstructured regions of these proteins become more ordered in response to their interaction with the nascent mineral particles.

Orchestrated regulation of iron trafficking proteins in the kidney during iron overload facilitates systemic iron retention.

The exact route of iron through the kidney and its regulation during iron overload are not completely elucidated. Under physiologic conditions, non-transferrin and transferrin bound iron passes the glomerular filter and is reabsorbed through kidney epithelial cells, so that hardly any iron is found in the urine. To study the route of iron reabsorption through the kidney, we analyzed the location and regulation of iron metabolism related proteins in kidneys of mice with iron overload, elicited by iron dextran injections. Transferrin Receptor 1 was decreased as expected, following iron overload. In contrast, the multi-ligand hetero-dimeric receptor-complex megalin/cubilin, which also mediates the internalization of transferrin, was highly up-regulated. Moreover, with increasing iron, intracellular ferritin distribution shifted in renal epithelium from an apical location to a punctate distribution throughout the epithelial cells. In addition, in contrast to many other tissues, the iron exporter ferroportin was not reduced by iron overload in the kidney. Iron accumulated mainly in interstitial macrophages, and more prominently in the medulla than in the cortex. This suggests that despite the reduction of Transferrin Receptor 1, alternative pathways may effectively mediate re-absorption of iron that cycles through the kidney during parenterally induced iron-overload. The most iron consuming process of the body, erythropoiesis, is regulated by the renal erythropoietin producing cells in kidney interstitium. We propose, that the efficient re-absorption of iron by the kidney, also during iron overload enables these cells to sense systemic iron and regulate its usage based on the systemic iron state.

Ferritin is secreted via 2 distinct nonclassical vesicular pathways.

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum-Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body-exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.

Long-acting glucose-dependent insulinotropic polypeptide ameliorates obesity-induced adipose tissue inflammation.

Obesity induces low-grade chronic inflammation, manifested by proinflammatory polarization of adipose tissue innate and adaptive resident and recruited immune cells that contribute to insulin resistance (IR). The glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that mediates postprandial insulin secretion and has anabolic effects on the adipose tissue. Importantly, recent evidence suggested that GIP is a potential suppressor of inflammation in several metabolic models. In this study, we aimed to investigate the immunoregulatory role of GIP in a murine model of diet-induced obesity (DIO) using the long-acting GIP analog [d-Ala(2)]GIP. Administration of [d-Ala(2)]GIP resulted in adipocytes of increased size, increased levels of adipose tissue lipid droplet proteins, indicating better lipid storage capacity, and reduced adipose tissue inflammation. Flow cytometry analysis revealed reduced numbers of inflammatory Ly6C(hi) monocytes and F4/80(hi)CD11c(+) macrophages, associated with IR. In addition, [d-Ala(2)]GIP reduced adipose tissue infiltration of IFN-γ-producing CD8(+) and CD4(+) T cells. Furthermore, [d-Ala(2)]GIP treatment induced a favorable adipose tissue adipokine profile, manifested by a prominent reduction in key inflammatory cytokines (TNF-α, IL-1ß, IFN-γ) and chemokines (CCL2, CCL8, and CCL5) and an increase in adiponectin. Notably, [d-Ala(2)]GIP also reduced the numbers of circulating neutrophils and proinflammatory Ly6C(hi) monocytes in mice fed regular chow or a high-fat diet. Finally, the beneficial immune-associated effects were accompanied by amelioration of IR and improved insulin signaling in liver and adipose tissue. Collectively, our results describe key beneficial immunoregulatory properties for GIP in DIO and reveal that its augmentation ameliorates adipose tissue inflammation and improves IR.

Differential stimulation of peripheral blood mononuclear cells in Crohn's disease by fungal glycans.

BACKGROUND AND AIM: Crohn's disease (CD) is characterized by loss of tolerance to intestinal microorganisms. This is reflected by serological responses to fungal glycans such as mannan and ß-glucans. Fungal glycans have various effects on immune cells. However, the evidence for their effects in CD is vague. This study aimed to assess the effects of fungal cell wall glycans on human peripheral blood mononuclear cells (PBMCs) from CD and control patients. METHODS: Human PBMCs from CD and control patients were stimulated by fungal cell wall glycans. Cytokine secretion was detected by ELISA and glycan receptor expression by flow cytometry. RESULTS: Mannan, ß-glucans (curdlan), chitosan, and zymosan induced the secretion of interleukin (IL)-1ß, IL-6, IL-23, IL-10, and tumor necrosis factor-α by PBMCs. Spleen tyrosin kinase and Src tyrosine kinase were involved in the response to mannan and ß-glucans. Mannan and whole yeast cells induced a significantly higher pro-inflammatory cytokine response in CD compared with control patients. CONCLUSIONS: The results may suggest that CD is characterized by hyperresponsiveness to fungal glycans. Thus, glycans may potentially be triggering or perpetuating inflammation.

Role of glucose-dependent insulinotropic polypeptide in adipose tissue inflammation of dipeptidylpeptidase 4-deficient rats.

OBJECTIVES: Dipeptidyl peptidase 4 (DPP4) inhibitors, used in obese diabetic patients, reduce inflammation in several models. The role of chronic DPP4-deficiency (DPP4-) in diet-induced obesity with respect to insulin sensitivity and adipose tissue inflammation was investigated. DESIGN AND METHODS: Insulin resistance was induced by 2 months high fat diet (HFD). In vitro effects of glucose-dependent insulinotropic polypeptide (GIP) were assessed in adipose tissue explants and stromal vascular fraction (SVF). RESULTS: HFD-fed DPP4-rats gained significantly more weight and visceral fat mass, yet were more insulin sensitive. Adipose tissue of DPP4- rats demonstrated increased adipocyte maturation and increased expression of enzymes involved in triglyceride uptake and synthesis, yet increased adiponectin mRNA, reduced mRNA of proinflammatory cytokines and reduced vascular adhesion molecules, suggesting reduced inflammation. In vitro and in vivo experiments explored the role of GIP in inducing this phenotype. Indeed, we demonstrated that GIP directly enhanced adiponectin expression in rat and human adipose tissue explants and in SVF. Lastly, GIP administration to normal or HFD-fed rats elevated serum adiponectin and improved their glucose tolerance test. CONCLUSION: In a HFD model, DPP4-rats exhibited reduced adipose tissue inflammation and improved insulin resistance, which may be mediated in part by GIP induction of adiponectin.