Characterization of Mutant Neutrophil Elastase in Severe Congenital Neutropenia

Li, Feng-Qian; Horwitz, Marshall S.

doi:10.1074/jbc.m010279200

Cited by 94 publications

(91 citation statements)

References 41 publications

(35 reference statements)

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“…However, the pattern of mutation in the neutrophil elastase gene differs in the 2 disorders, 26 suggesting that the mutated enzyme acts by a different mechanism in each disease. Mutated elastase complexes poorly with ␣-1 antitrypsin 27 and may be less sensitive to other serpins. This raises the possibility that neutropenia could be a consequence of increased destruction of G-CSF by more abundant elastase.…”

Section: Discussionmentioning

confidence: 99%

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis

Ouriaghli

Fujiwara

Melenhorst

et al. 2003

Blood

100

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There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth, and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3, elastase, azurocidin, and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34 ؉ cells, elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase, G-CSF, but not GM-CSF or SCF, was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of

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Section: Discussionmentioning

confidence: 99%

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis

Ouriaghli

Fujiwara

Melenhorst

et al. 2003

Blood

100

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“…This network of interactions between N, NusA, and boxB, followed by the interactions between NusE, NusB, and the boxA RNA sequence and then the interactions of N, NusA, NusE, and NusG with RNA polymerase (22,48,49,51), creates a complex network that is stable and active during transcription over thousands of nucleotide residues and enables expression of the phage late genes during the switch from lysogeny to lysis. The 21 and 22 boxA sequences are identical to the rrn boxA sequence, and so NusE is likely to bind to this NusB-RNA complex at lower concentrations than the free protein concentration at which it binds to the boxA sequences.…”

Section: In Vitro Assembly Of the Central Components Of The Boxa-depementioning

confidence: 99%

Assembly of an RNA-Protein Complex

Greive

Lins²,

Hippel

2005

Journal of Biological Chemistry

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Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K d values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K d values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available singlestranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.Macromolecular machines involving multiple protein and nucleic acid components drive and regulate many important cellular processes. These include gene expression, protein synthesis and trafficking, as well as signal transduction and many other processes. The assembly of the relevant macromolecular complexes is often controlled by additional regulatory factors that modulate the effective binding affinity of the central components of a self-assembling macromolecular machine. Another regulatory strategy involves controlling the local concentration of a critical component of the complex, which can perturb crucial binding interactions by manipulating mass action effects. In principle this strategy uses changes in the concentration parameter as a "switch" to turn the targeted assembly reaction on or off; a good example of this strategy involves using the control of the synthesis (and thus of the free concentration) of the single-stranded DNA-binding protein of T4 (gp32) to regulate the assembly of the functional T4 DNA replication complex (1). Furthermore, if this component is "tethered" in the vicinity of the assembly to be regulated (e.g. by cis-RNA looping (2, 3)), this switch can be used to confine the regulation of concentration change only to nearby target loci on the genome and not to others.Characterizing the binding interactions between particular components of a macromolecular complex and elucidating the mechanisms of assembly is a challenging, but vital, part of understanding how cellular processes are regulated. This problem is compounded when...

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“…We measured neutrophil elastase activity in extracts of 10 5 mononuclear cells from freshly drawn peripheral blood purified on Ficoll-Hypaque density gradients spectrophotometrically on the specific substrate suc-Ala-Ala-Ala-pNa as described 15 .…”

Section: Neutrophil Elastase Activity Assaymentioning

confidence: 99%

“…These ELA2 mutations do not consistently abrogate proteolytic activity, and their biochemical consequences are unclear 15 . Most commonly, these mutations delete the C-terminal extension of neutrophil elastase.…”

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confidence: 99%

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

et al. 2003

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Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic 1,2 . We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.Low neutrophil numbers lead to opportunistic infections. There are two hereditary human neutropenia syndromes: cyclic hematopoiesis 4 , comprising three-week oscillations of blood cells, and SCN 3 , consisting of statically low neutrophil counts progressing to leukemia. Heterozygous mutations of ELA2 cause cyclic hematopoiesis and about two-thirds of SCN cases. Mutations in WAS (different from those that cause Wiskott-Aldrich thrombocytopenia) also cause SCN 5 . Owing to its severity, SCN usually arises from new mutations, and additional genes associated with neutropenia have not yet been identified.

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Characterization of Mutant Neutrophil Elastase in Severe Congenital Neutropenia

Cited by 94 publications

References 41 publications

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis

Assembly of an RNA-Protein Complex

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

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