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Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (␤ 2 -microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-Cspecific motif KYRV at residues 66 -76 of the ␣1-domain ␣-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locusspecific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.Class I human leukocyte antigens (called HLA) are cell-surface heterotrimers formed by a highly polymorphic heavy (44 kDa) chain, a non-polymorphic light (12 kDa) chain subunit (␤ 2 -microglobulin (␤ 2 m)3 ), and a short (8 -11-mer) peptide antigen derived from the degradation of intracellular proteins (1). The assembly pathway of most class I molecules involves an early interaction of the heavy chain, still free of ␤ 2 m, with calnexin, followed by association with ␤ 2 m and binding to the so-called peptide-loading complex. This is a supramolecular endoplasmic reticulum structure comprising, among others, two HLA-dedicated chaperones: TAP transporter associated with antigen processing) and the peptide editor/facilitator tapasin (1). Successful peptide loading results in tight association of the heavy chain with ␤ 2 m and the release of thermally stable, folded class I conformers (1-3). These are exported to the cell surface, where they activate and inhibit cytotoxic T lymphocytes expressing the rearranging T cell receptor and natural killer (NK) cells expressing non-rearranging receptors such as the killer immunoglobulin-like receptors, respectively (4).There are Ͼ1000 class I molecules, encoded by three highly polymorphic allelic series: HLA-A, -B, and -C (www. anthonynolan.org.UK/HIG/index.html). They share a conserved general architecture, a common peptide-loading pathway, and a similar set of functions, but also display a number of allel...

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Class I MHC H chains assemble with β2-microglobulin (β2m) and are loaded with peptide Ags through multiple folding steps. When free of β2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, β2m-free H chains with calnexin in β2m-defective cells, and with calreticulin and TAP in β2m-expressing cells. In β2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the α1 domain α helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the α1 domain α helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.

The growing use of antimony (Sb) compounds in industry and the consequent increase in the number of exposed workers make it important to carry out a health risk assessment. The main goal of this study was to assess the genotoxicity of Sb(2)O(3) in occupationally exposed workers. Genotoxicity was evaluated by the sister chromatid exchange (SCE) and micronucleus tests, and the enzyme (Fpg)-modified comet assay. In addition, antimony exposure levels were established by environmental monitoring with personal air samplers. We studied 23 male workers assigned to different fire retardant treatment tasks in the car upholstery industry and a control group of 23 healthy nonexposed males. The exposed workers were divided into two groups on the basis of their tasks and the work cycle: Group A comprised finishing and intermediate inspection operators who directly handled a mixture containing Sb(2)O(3); Group B were jet operators, not directly exposed to the compound. Environmental monitoring detected low Sb exposure levels but significant differences between the two groups, with Group A having the higher exposure level. Cytogenetic analyses showed no difference between exposed workers and controls for micronuclei and SCE. The enzyme-modified comet assay showed a probable relation between moderate levels of oxidative DNA damage and exposure to antimony, with a significantly higher proportion of workers in Group A having oxidative DNA damage compared to controls. The results support the theory that oxidative DNA damage is involved in the genotoxicity of antimony and indicate the need for further research in this field.

Chemical chaperonesp reventprotein aggregation. However,t he use of chemicalc haperones as drugs against diseases due to protein aggregation is limitedb yt he very high active concentrations (mm range) required to mediate their effect. One of the most commonc hemical chaperones is 4-phenylbutyric acid (4-PBA). Despite itsu nfavorablep harmacokinetic properties, 4-PBA was approved as ad rug to treat ornithine cycled iseases.H ere, we report that 2-isopropyl-4-phenylbutanoic acid (5)h as been found to be 2-10fold more effective than 4-PBA in several in vitro modelso f protein aggregation.I mportantly,c ompound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451CN LGN3).[a] S. Azoulay-Ginsburg,L .L evy,P rof. A. Gruzman