Rikke Nielsen

Incorporation of Aminoacyl-tRNA into the Ribosome as seen by Cryo-electron Microscopy

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of ∼9 Å, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.

Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules

Loss of the renal endosome-associated chloride channel, ClC-5, in Dents disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected125I-β2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

Receptor-mediated endocytosis in renal proximal tubule.

Proteins filtered in renal glomeruli are removed from the tubular fluid by endocytosis in the proximal tubule mediated by the two receptors megalin and cubilin. After endocytic uptake, the proteins are transferred to lysosomes for degradation, while the receptors are returned to the apical cell membrane by receptor recycling in dense apical tubules. In the renal proximal tubule, there is no significant transcellular transport of protein. The reabsorptive process is extremely efficient as evidenced by the virtual protein free urine in humans. The two receptors bind a variety of ligands. The process serves not only to remove the proteins from the ultrafiltrate but also to conserve a variety of essential substances such as vitamins and trace elements carried by plasma proteins. The endocytic apparatus is highly developed in the proximal tubule demonstrating the high capacity of the cells; however, under certain circumstances like diseases affecting the glomeruli, the system is overloaded resulting in proteinuria.

Endocytic Receptors in the Renal Proximal Tubule

Protein reabsorption is a predominant feature of the renal proximal tubule. Animal studies show that the ability to rescue plasma proteins relies on the endocytic receptors megalin and cubilin. Recently, studies of patients with syndromes caused by dysfunctional receptors have supported the importance of these for protein clearance of human ultrafiltrate. This review focuses on the molecular biology and physiology of the receptors and their involvement in renal pathological conditions.

Megalin Antagonizes Activation of the Parathyroid Hormone Receptor

Parathyroid hormone (PTH) is predominantly cleared from the circulation by glomerular filtration and degradation in the renal proximal tubules. Here, we demonstrate that megalin, a multifunctional endocytic receptor in the proximal tubular epithelium, mediates the uptake and degradation of PTH. Megalin was purified from kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH fragments (K d 0.5 μm). Absence of the receptor in megalin knockout mice resulted in 4-fold increased levels of amino-terminal PTH fragments in the urine. In F9 cells expressing both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP receptor), uptake and lysosomal degradation of the hormone was mediated through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data provide evidence that megalin is involved in the renal catabolism of PTH and potentially antagonizes PTH/PTHrP receptor activity in the proximal tubular epithelium.

Listeria monocytogenes induces IFNβ expression through an IFI16‐, cGAS‐ and STING‐dependent pathway

Listeria monocytogenes is a gram‐positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon β (IFNβ) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria‐derived cyclic‐di‐AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNβ expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic‐di‐AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNβ expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.

Transforming growth factor‐β1 reduces megalin‐ and cubilin‐mediated endocytosis of albumin in proximal‐tubule‐derived opossum kidney cells

Transforming growth factor (TGF)‐β1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial‐mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF‐β1‐induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin‐cubilin‐dependent receptor‐mediated endocytosis. In this study we used a well established cell culture model (proximal‐tubule‐derived opossum kidney (OK) cells) in order to test the hypothesis that TGF‐β1 reduces megalin‐cubilin‐mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF‐β1 led to a time‐ and dose‐dependent downregulation of megalin‐cubilin‐mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF‐β1 action does not involve mitogen‐activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF‐β1‐induced downregulation of megalin‐cubilin‐mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF‐β1. In conclusion our data indicate that enhanced levels of TGF‐β1 occurring in various nephropathies can lead to downregulation of megalin‐cubilin‐dependent endocytosis. Probably, TGF‐β1 leads to Smad2‐ and Smad3‐dependent expression of negative regulators of receptor‐mediated endocytosis.

Proteinuria and events beyond the slit

The origin of proteinuria is found in either the glomerular filtration device or the proximal tubular reabsorption machinery. During equilibrium, small amounts of predominantly low molecular weight proteins are filtered and reabsorbed by the receptor complex megalin/cubilin/amnionless. This results in a protein-free filtrate passing further down the tubule. During glomerular damage, the reabsorption machinery in the proximal tubule is challenged due to elevated amounts of proteins passing the glomerular filtration slits. Even though it is considered to be a high-capacity system, several conditions result in proteinuria, thus exposing the cells in the rest of the nephron to a protein-rich environment. The impact on cells in the more distal part of the nephron is uncertain, but studies support an involvement in fibrosis development. Protein accumulation in lysosomes of the proximal tubule, due to increased protein internalization, is thought to mediate inflammation and fibrosis, eventually leading to renal failure. In contrast, low molecular weight proteinuria develops when the endocytic machinery is malfunctioning either by direct or indirect causes such as in Imerslund-Gräsbeck syndrome (IGS) or Dent’s disease, respectively. This review discusses the origin of proteinuria and describes the structural fundament for protein reabsorption in the proximal tubule as well as conditions resulting in low molecular weight proteinuria.

Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs.

Megalin and cubilin in proximal tubule protein reabsorption: from experimental models to human disease

Proximal tubule protein uptake is mediated by 2 receptors, megalin and cubilin. These receptors rescue a variety of filtered ligands, including biomarkers, essential vitamins, and hormones. Receptor gene knockout animal models have identified important functions of the receptors and have established their essential role in modulating urinary protein excretion. Rare genetic syndromes associated with dysfunction of these receptors have been identified and characterized, providing additional information on the importance of these receptors in humans. Using various disease models in combination with receptor gene knockout, the implications of receptor dysfunction in acute and chronic kidney injury have been explored and have pointed to potential new roles of these receptors. Based on data from animal models, this paper will review current knowledge on proximal tubule endocytic receptor function and regulation, and their role in renal development, protein reabsorption, albumin uptake, and normal renal physiology. These findings have implications for the pathophysiology and diagnosis of proteinuric renal diseases. We will examine the limitations of the different models and compare the findings to phenotypic observations in inherited human disorders associated with receptor dysfunction. Furthermore, evidence from receptor knockout mouse models as well as human observations suggesting a role of protein receptors for renal disease will be discussed in light of conditions such as chronic kidney disease, diabetes, and hypertension.