Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Neutralization of pro‐inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C⁺ monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C^hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.     

The human islet amyloid polypeptide forms transient membrane-active prefibrillar assemblies

The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.     

GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28

Membrane proteins located on vesicles (v-SNAREs) and on the target membrane (t-SNAREs) mediate specific recognition and, possibly, fusion between a transport vesicle and its target membrane. The activity of SNARE molecules is regulated by several soluble cytosolic proteins. We have cloned a bovine brain cDNA encoding a conserved 117 amino acid polypeptide, denoted Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), that functions as a soluble transport factor. GATE-16 interacts with N-ethylmaleimidesensitive factor (NSF) and significantly stimulates its ATPase activity. It also interacts with the Golgi v-SNARE GOS-28 in an NSF-dependent manner. We propose that GATE-16 modulates intra-Golgi transport through coupling between NSF activity and SNAREs activation.     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

The membrane-embedded domain of the unusual electron transporter DsbD (DsbDbeta) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDbeta displays an inherent functional and structural symmetry: first, the two cysteines of DsbDbeta can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDbeta and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins.