Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform ␤-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for ␤-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to ␤-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for ␤-tubulin and not for ␣-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated ␤-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of ␤-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated ␤-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a ␤-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.Microtubules have important functions in a variety of dynamic activities within the cell including intracellular transport, cell motility, and cell division. They provide structural support for the cell and are an important component of the cytoskeletal framework that generates cell morphology. In neurons microtubules are required for formation of specialized processes, i.e. dendrites and axons. Structural microtubule-associated proteins (MAPs), 2 such as MAP2 and tau, regulate the stability of microtubules in those processes.Microtubules are predominantly composed of tubes of polymerized dimers of ␣-and ␤-tubulin. A central question in cell biology is how functional heterogeneity is imparted to different types (e.g. dendritic, axonal, and centriolar) of microtubules. Two basic mechanisms have been proposed to explain this issue. The first involves duplication and divergence of genes encoding tubulin. The second mechanism involves the addition of a variety of post-translational modifications (PTM) to microtubules that further increases the tubulin heterogeneity (1). These PTMs are asso...

Tubular injury and interstitial fibrosis are the hallmarks of chronic kidney disease. While recent studies have verified that proximal tubular injury triggers interstitial fibrosis, the impact of fibrosis on tubular injury and regeneration remains poorly understood. We generated a novel mouse model expressing diphtheria toxin receptor on renal fibroblasts to allow for the selective disruption of renal fibroblast function. Administration of diphtheria toxin induced upregulation of the tubular injury marker Ngal and caused tubular proliferation in healthy kidneys, whereas administration of diphtheria toxin attenuated tubular regeneration in fibrotic kidneys. Microarray analysis revealed down-regulation of the retinol biosynthesis pathway in diphtheria toxin-treated kidneys. Healthy proximal tubules expressed retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in retinoic acid biosynthesis. After injury, proximal tubules lost RALDH2 expression, whereas renal fibroblasts acquired strong expression of RALDH2 during the transition to myofibroblasts in several models of kidney injury. The retinoic acid receptor (RAR) RARg was expressed in proximal tubules both with and without injury, and aBcrystallin, the product of an RAR target gene, was strongly expressed in proximal tubules after injury. Furthermore, BMS493, an inverse agonist of RARs, significantly attenuated tubular proliferation in vitro. In human biopsy tissue from patients with IgA nephropathy, detection of RALDH2 in the interstitium correlated with older age and lower kidney function. These results suggest a role of retinoic acid signaling and cross-talk between fibroblasts and tubular epithelial cells during tubular injury and regeneration, and may suggest a beneficial effect of fibrosis in the early response to injury.Kidney International (2019) 95, 526-539; https://doi.

Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of ␣-(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-D-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.The significance of the muscle and liver in amino acid metabolism has been well recognized and the involvement of these two tissues in amino acid uptake has been investigated in detail (1). The adipose tissue also possesses multiple amino acid transport systems (2). In addition to the obvious role of amino acids in protein synthesis, available evidence indicates that amino acids may have other important functions in the adipose tissue. Adipose tissue uses amino acids to synthesize fatty acids, triglycerides, and glycerol-based phospholipids (3, 4). Furthermore, amino acids modulate the magnitude of insulin-stimulated translocation of the facilitated glucose transporter GLUT4 (5), glutamine modulates fat metabolism through a regulatory effect on hexosamine biosynthesis (6), and arginine enhances insulin-stimulated glycogen synthesis (7). Therefore, it seems that amino acid uptake may influence glucose and fat metabolism in adipose tissue.Under physiological conditions, uptake of small neutral and gluconeogenic amino acids such as glutamine and alanine into adipocytes occurs primarily via the amino acid transport system A (2). System A is a Na ϩ -dependent active transport system for neutral amino acids expressed in most tissues (8). A unique characteristic of this system is its ability to recognize N-alkylated amino acids as substrates (9). ␣-(Methylamino)isobutyric acid (MeAIB) 2 is commonly used as a model s...

Neutrophil gelatinase-associated lipocalin (NGAL, lipocalin 2 or LCN2) is an iron carrier protein whose circulating level is increased by kidney injury, bacterial infection and obesity, but its metabolic consequence remains elusive. To study physiological role of LCN2 in energy homeostasis, we challenged female Lcn2 knockout (KO) and wild-type (WT) mice with high fat diet (HFD) or cold exposure. Under normal diet, physical constitutions of Lcn2 KO and WT mice were indistinguishable. During HFD treatment, Lcn2 KO mice exhibited larger brown adipose tissues (BAT), consumed more oxygen, ate more food and gained less body weights as compared to WT mice. When exposed to 4 °C, KO mice showed higher body temperature and more intense 18F-fluorodeoxyglucose uptake in BAT, which were cancelled by β3 adrenergic receptor blocker or iron-loaded (but not iron-free) LCN2 administration. These findings suggest that circulating LCN2 possesses obesity-promoting and anti-thermogenic effects through inhibition of BAT activity in an iron-dependent manner.