Summary Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A4 hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B4. We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.
The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K d ؍ 30 -50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.point mutants ͉ secretion ͉ protein evolution N iemann-Pick Type C (NPC) is an autosomal-recessive disorder characterized by progressive ataxia, dystonia, and dementia (1). Substantial cell death, particularly in the cerebellum, accounts for some of the symptoms. At the cellular level, mutant cells accumulate cholesterol and other lipids in aberrant compartments with features of late endosomes and lysosomes, and the normal homeostatic response to this excess cholesterol is abolished (2-4).Our understanding of the molecular basis of this disease has progressed substantially over the last several years, because the two genes damaged by NPC mutations, NPC1 and NPC2, have been identified (5-7). NPC1 encodes a multiple-membranespanning protein containing sequences similar to the sterol cleavage activating protein (SCAP) regulator of cholesterol metabolism and the receptor Patched, a transducer of the Hedgehog family of protein signals. Mutations in NPC1 are responsible for Ϸ95% of the NPC cases (8). The exact role that NPC1 plays in maintaining normal levels of cholesterol in late endosomes and lysosomes remains mysterious. Even less is known about the NPC2 [human epididymis (HE1)] protein, the loss of which is responsible for 5% of NPC cases.NPC2 is a small secreted glycoprotein originally identified as a transcript enriched in the HE1 (9). The porcine HE1 homolog was reported to bind cholesterol with micromolar affinity (K d ϭ 2.3 M; ref. 10), information that was crucial in identifying HE1 as a candidate npc2 gene product (7). However, the similarity between the reported K d and the solubility of cholesterol in aqueous solution (5 M, Merck Index) warrants reexamination of the strength and specificity of this interaction. If the binding is meaningful, NPC2 activity could be altered when cholesterol is bound, thus serving as a sensor of cholesterol levels, accessibility, or location. Alternatively, NPC2 could process or transport cholesterol. Resolving these questions is crucial for determining why cholesterol accumulates in people lacking functional NPC2 protein and for designing rational treatment strategies.In this report, we combine in vitro binding experiments, genetic analysis, a quantitative cell-based ...
Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death.
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