Complex Inhibitory Mechanism of Glycomimetics with Heparanase.

Heparanase (HPSE) is the only mammalian endo-ß-glucuronidase known to catalyze the degradation of heparan sulfate. Dysfunction of HPSE activity has been linked to several disease states, resulting in HPSE becoming the target of numerous therapeutic programs, yet no drug has passed clinical trials to date. Pentosan polysulfate sodium (PPS) is a heterogeneous, FDA-approved drug for the treatment of interstitial cystitis and a known HPSE inhibitor. However, due to its heterogeneity, characterization of its mechanism of HPSE inhibition is challenging. Here, we show that inhibition of HPSE by PPS is complex, involving multiple overlapping binding events, each influenced by factors such as oligosaccharide length and inhibitor-induced changes in the protein secondary structure. The present work advances our molecular understanding of the inhibition of HPSE and will aid in the development of therapeutics for the treatment of a broad range of pathologies associated with enzyme dysfunction, including cancer, inflammatory disease, and viral infections.

A Rapid and Mild Sulfation Strategy Reveals Conformational Preferences in Therapeutically Relevant Sulfated Xylooligosaccharides.

Although sulfated xylooligosaccharides are promising therapeutic leads for a multitude of afflictions, the structural complexity and heterogeneity of commercially deployed forms (e. g. Pentosan polysulfate 1) complicates their path to further clinical development. We describe herein the synthesis of the largest homogeneous persulfated xylooligomers prepared to date, comprising up to eight xylose residues, as standards for biological studies. Near quantitative sulfation was accomplished using a remarkably mild and operationally simple protocol which avoids the need for high temperatures and a large excess of the sulfating reagent. Moreover, the sulfated xylooligomer standards so obtained enabled definitive identification of a pyridinium contaminant in a sample of a commercially prepared Pentosan drug and provided significant insights into the conformational preferences of the constituent persulfated monosaccharide residues. As the spatial distribution of sulfates is a key determinant of the binding of sulfated oligosaccharides to endogenous targets, these findings have broad implications for the advancement of Pentosan-based treatments.

Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells.

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains--an Ndfip1-YFP reporter and an Ndfip1-deficient strain--to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4(+) T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4(+) cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity.

A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration.

A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders.

Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.

BACKGROUND: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. METHODS AND FINDINGS: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis. CONCLUSIONS: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.

A role for Alström syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence.

Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alström syndrome. Alström syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alström syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.

Fat aussie--a new Alström syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis.

Mutations in the human ALMS1 gene are responsible for Alström syndrome, a disorder in which key metabolic and endocrinological features include childhood-onset obesity, metabolic syndrome, and diabetes, as well as infertility. ALMS1 localizes to the basal bodies of cilia and plays a role in intracellular trafficking, but the biological functions of ALMS1 and how these relate to the pathogenesis of obesity, diabetes, and infertility remain unclear. Here we describe a new mouse model of Alström syndrome, fat aussie, caused by a spontaneous mutation in the Alms1 gene. Fat aussie (Alms1 foz/foz) mice are of normal weight when young but, by 120 d of age, they become obese and hyperinsulinemic. Diabetes develops in Alms1 foz/foz mice accompanied by pancreatic islet hyperplasia and islet cysts. Female mice are fertile before the onset of obesity and metabolic syndrome; however, male fat aussie mice are sterile due to a progressive germ cell loss followed by an almost complete block of development at the round-to-elongating spermatid stage of spermatogenesis. In conclusion, Alms1 foz/foz mouse is a new animal model in which to study the pathogenesis of the metabolic and fertility defects of Alström syndrome, including the role of ALMS1 in appetite regulation, pathogenesis of the metabolic syndrome, pancreatic islet physiology, and spermatogenesis.

Widespread failure of hematolymphoid differentiation caused by a recessive niche-filling allele of the Ikaros transcription factor.

A central issue in understanding the hematolymphoid system is the generation of appropriate mutant alleles in mice to reveal the function of regulatory genes. Here we describe a mouse strain, Plastic, with a point mutation in a zinc finger of Ikaros that disrupts DNA binding but preserves efficient assembly of the full-length protein into higher order complexes. Ikaros(Plastic) homozygosity is embryonically lethal with severe defects in terminal erythrocyte and granulocyte differentiation, excessive macrophage formation, and blocked lymphopoiesis, while heterozygotes display a partial block in lymphocyte differentiation. The contrast with more circumscribed effects of Ikaros alleles that ablate the full-length protein highlights the importance in mammals of generating recessive niche-filling alleles that inactivate function without creating a void in multimolecular assemblies.

Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis.

In a genome-wide ENU mouse mutagenesis screen a recessive mouse mutation, unmodulated, was isolated with profound defects in humoral immune responses, selective deficits in B cell activation by antigen receptors and T cell costimulation by CD28, and gradual development of atopic dermatitis with hyper-IgE. Mutant B cells are specifically defective in forming connections between antigen receptors and two key signaling pathways for immunogenic responses, NF-kappaB and JNK, but signal normally to calcium, NFAT, and ERK. The mutation alters a conserved leucine in the coiled-coil domain of CARMA-1/CARD11, a member of the MAGUK protein family implicated in organizing multimolecular signaling complexes. These results define Carma-1 as a key regulator of the plasticity in antigen receptor signaling that underpins opposing mechanisms of immunity and tolerance.

Contribution of the Box 1 and Box 2 motifs of cytokine receptors to Jak1 association and activation.

Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the beta chain of type I interferon receptor (IFNalphaRbetaL/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1. On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor beta (IL2Rbeta), IFNgammaRalpha/IFNGR1, and IL10Ralpha/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jak1. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFNalphaRbetaL/IFNAR2 and IL2Rbeta subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFNgammaRalpha/IFNGR1 (SILLPKS) and IL10Ralpha/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8-10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38-58 amino acids from the membrane).