Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

Elevated Serum Amino Acids Induce a Subpopulation of Alpha Cells to Initiate Pancreatic Neuroendocrine Tumor Formation.

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing ß cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Comprehensive genomic analysis identifies pathogenic variants in maturity-onset diabetes of the young (MODY) patients in South India.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.

Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels.

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

Amelioration of type 2 diabetes by antibody-mediated activation of fibroblast growth factor receptor 1.

Clinical use of recombinant fibroblast growth factor 21 (FGF21) for the treatment of type 2 diabetes and other disorders linked to obesity has been proposed; however, its clinical development has been challenging owing to its poor pharmacokinetics. Here, we describe an alternative antidiabetic strategy using agonistic anti-FGFR1 (FGF receptor 1) antibodies (R1MAbs) that mimic the metabolic effects of FGF21. A single injection of R1MAb into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, along with marked improvement in hyperinsulinemia, hyperlipidemia, and hepatosteatosis. R1MAb activated the mitogen-activated protein kinase pathway in adipose tissues, but not in liver, and neither FGF21 nor R1MAb improved glucose clearance in lipoatrophic mice, which suggests that adipose tissues played a central role in the observed metabolic effects. In brown adipose tissues, both FGF21 and R1MAb induced phosphorylation of CREB (cyclic adenosine 5'-monophosphate response element-binding protein), and mRNA expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α) and the downstream genes associated with oxidative metabolism. Collectively, we propose FGFR1 in adipose tissues as a major functional receptor for FGF21, as an upstream regulator of PGC-1α, and as a compelling target for antibody-based therapy for type 2 diabetes and other obesity-associated disorders.

Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3-null embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

FGF19 regulates cell proliferation, glucose and bile acid metabolism via FGFR4-dependent and independent pathways.

Fibroblast growth factor 19 (FGF19) is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF19 also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF19 activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4), although FGF19 can activate other FGFRs in vitro in the presence of the coreceptor ßKlotho (KLB). In this report, we investigate the role of FGFR4 in mediating FGF19 activity by using Fgfr4 deficient mice as well as a variant of FGF19 protein (FGF19v) which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF19, but is not essential for FGF19 to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF19 acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation.

Functional characterization of putative cilia genes by high-content analysis.

Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates.

LRP2 in ependymal cells regulates BMP signaling in the adult neurogenic niche.

The microenvironment of growth factors in the subependymal zone (SEZ) of the adult brain provides the instructive milieu for neurogenesis to proceed in this germinal niche. In particular, tight regulation of bone morphogenetic protein (BMP) signaling is essential to balance proliferative and non-proliferative cell fate specification. However, the regulatory pathways that control BMP signaling in the SEZ are still poorly defined. We demonstrate that LRP2, a clearance receptor for BMP4 is specifically expressed in ependymal cells of the lateral ventricles in the adult brain. Intriguingly, expression is restricted to the ependyma that faces the stem cell niche. Expression is not seen in ependyma elsewhere in the lateral ventricles or in the dentate gyrus, the second major neurogenic zone of the adult brain. We further show that lack of LRP2 expression in adult mice results in impaired proliferation of neural precursor cells in the SEZ resulting in decreased numbers of neuroblasts reaching the olfactory bulb. Reduced neurogenesis coincides with increased BMP4 expression and enhanced activation of downstream mediators phospho-SMAD1/5/8 and ID3 in the stem cell niche. Our findings suggest a novel mechanism whereby LRP2-mediated catabolism of BMP4 in the ependyma modulates the microenvironment of the SEZ and enables adult neurogenesis to proceed.

Wnt signaling is regulated by endoplasmic reticulum retention.

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.

The mammalian Cos2 homolog Kif7 plays an essential role in modulating Hh signal transduction during development.

The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, we demonstrate that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. We show that Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. We also demonstrate a requirement for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh.

Kinome siRNA screen identifies regulators of ciliogenesis and hedgehog signal transduction.

Disruption or improper activation of the Hedgehog (Hh) pathway is associated with developmental abnormalities and cancer. Although characterized in Drosophila, the mechanisms that mediate the Hh signal downstream of the Smoothened (Smo) seven-transmembrane protein in vertebrates remain poorly understood. In particular, the Fused (Fu) kinase, which mediates Hh signaling in flies, is dispensable in mammals. To identify kinases that positively regulate the Hh pathway in mammals, we screened a mouse kinome small interfering RNA library and validated nine candidates that modulate Hh signaling. Among these candidates, Nek1 and Prkra did not directly function in the Hh pathway but exerted their effects on Hh signaling indirectly through a primary role in ciliogenesis. In contrast, another kinase, Cdc2l1, directly participated in the Hh pathway. Cdc2l1 was necessary and sufficient for activation of the Hh pathway, functioning downstream of Smo and upstream of the Glioma-associated (Gli) transcription factors. More specifically, Cdc2l1 interacted with the negative regulator Suppressor of Fused (Sufu) and relieved its inhibition on Gli, thus providing a mechanism for how Cdc2l1 might play a role in Hh signaling. Finally, with zebrafish as model organism, we showed that Cdc2l1 activated the Hh pathway in vivo. We propose that Cdc2l1 is a previously unrecognized member of the Hh signal transduction cascade.

Cortical dysplasia and skull defects in mice with a Foxc1 allele reveal the role of meningeal differentiation in regulating cortical development.

We report the identification of a hypomorphic mouse allele for Foxc1 (Foxc1(hith)) that survives into adulthood revealing previously unknown roles for Foxc1 in development of the skull and cerebral cortex. This line of mice was recovered in a forward genetic screen using ENU mutagenesis to identify mutants with cortical defects. In the hith allele a missense mutation substitutes a Leu for a conserved Phe at amino acid 107, leading to destabilization of the protein without substantially altering transcriptional activity. Embryonic and postnatal histological analyses indicate that diminished Foxc1 protein expression in all three layers of meningeal cells in Foxc1(hith/hith) mice contributes to the cortical and skull defects in mutant mice and that the prominent phenotypes appear as the meninges differentiate into pia, arachnoid, and dura. Careful analysis of the cortical phenotypes shows that Foxc1(hith/hith) mice display detachment of radial glial endfeet, marginal zone heterotopias, and cortical dyslamination. These abnormalities have some features resembling defects in type 2 (cobblestone) lissencephaly or congenital muscular dystrophies but appear later in corticogenesis because of the delay in breakdown of the basement membrane. Our data reveal that the meninges regulate the development of the skull and cerebral cortex by controlling aspects of the formation of these neighboring structures. Furthermore, we provide evidence that defects in meningeal differentiation can lead to severe cortical dysplasia.

Functional architecture of atrophins.

Vertebrate genomes harbor two Atrophin genes, Atrophin-1 (Atn1) and Atrophin-2 (Atn2). The Atn1 locus produces a single polypeptide, whereas two different protein products are expressed from the Atn2 (also known as Rere) locus. A long, or full-length, form contains an amino-terminal MTA-2-homologous domain followed by an Atrophin-1-related domain. A short form, expressed via an internal promoter, consists solely of the Atrophin domain. Atrophin-1 can be co-immunoprecipitated along with Atrophin-2, suggesting that the Atrophins ordinarily function together. Mutations that disrupt the expression of the long form of Atrophin-2 disrupt early embryonic development. To determine the requirement for Atrophin-1 during development we generated a null allele. Somewhat surprisingly we found that Atrophin-1 function is dispensable. To gain a better understanding of the requirement for Atrophin function during development, an analysis of the functional domains of the three different gene products was carried out. Taken together, these data suggest that Atrophins function as bifunctional transcriptional regulators. The long form of Atrophin-2 has a transcriptional repression activity that is not found in the other Atrophin polypeptides and that is required for normal embryogenesis. Atrophin-1 and the short form of Atrophin-2, on the other hand, can act as potent and evolutionarily conserved transcriptional activators.

Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli.

Sonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. Here, we show that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, we show that the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein.

A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development.

Although the mechanisms that regulate development of the cerebral cortex have begun to emerge, in large part through the analysis of mutant mice (Boncinelli et al. 2000; Molnar and Hannan 2000; Walsh and Goffinet 2000), many questions remain unanswered. To provide resources for further dissecting cortical development, we have carried out a focused screen for recessive mutations that disrupt cortical development. One aim of the screen was to identify mutants that disrupt the tangential migration of interneurons into the cortex. At the same time, we also screened for mutations that altered the growth or morphology of the cerebral cortex. We report here the identification of thirteen mutants with defects in aspects of cortical development ranging from the establishment of epithelial polarity to the invasion of thalamocortical axons. Among the collection are three novel alleles of genes for which mutant alleles had already been used to explore forebrain development, and four mutants with defects in interneuron migration. The mutants that we describe here will aid in deciphering the molecules and mechanisms that regulate cortical development. Our results also highlight the utility of focused screens in the mouse, in addition to the large-scale and broadly targeted screens that are being carried out at mutagenesis centers.

A Titin mutation defines roles for circulation in endothelial morphogenesis.

Morphogenesis of the developing vascular network requires coordinated regulation of an extensive array of endothelial cell behaviors. Precisely regulated signaling molecules such as vascular endothelial growth factor (VEGF) direct some of these endothelial behaviors. Newly forming blood vessels also become subjected to novel biomechanical forces upon initiation of cardiac contractions. We report here the identification of a recessive mouse mutation termed shrunken-head (shru) that disrupts function of the Titin gene. Titin was found to be required for the initiation of proper heart contractions as well as for maintaining the correct overall shape and orientation of individual cardiomyocytes. Cardiac dysfunction in shrunken-head mutant embryos provided an opportunity to study the effects of lack of blood circulation on the morphogenesis of endothelial cells. Without blood flow, differentiating endothelial cells display defects in their shapes and patterns of cell-cell contact. These endothelial cells, without exposure to blood circulation, have an abnormal distribution within vasculogenic vessels. Further effects of absent blood flow include abnormal spatial regulation of angiogenesis and elevated VEGF signaling. The shrunken-head mutation has provided an in vivo model to precisely define the roles of circulation on cellular and network aspects of vascular morphogenesis.