SummaryPresentation of antigenic peptides by human leukocyte antigen class I molecules is dependent on peptide transport into the endoplasmic reticulum by the transporters associated with antigen processing (TAP) (Germain, R. N. 1994. Cell. 76:287-299) . This translocation step is currently regarded as permissive for all peptides with COON-terminal residues capable of binding to HLA class I molecules (Momburg, F., J . Roelse, J. C. Howard, G. W. Butcher, G . J. Hammerling, and J. J. Neefjes . 1994. Nature (Loud .) . 367 :648-651) . In this report, we show that the human transporter selects peptides according to a binding motif based on the strong effects on peptide affinity of the three NHZ-terminal positions and the COOH-terminal residues . TAP favors strongly hydrophobic residues in position 3 (P3) and hydrophobic or charged residues in P2, whereas aromatic or acidic residues in P1, as well as Pro in P1 and P2, have strong deleterious effects . Selection of naturally presented peptides by the transporter is suggested by their higher average affinity for TAP, as compared to nonselected peptides . The TAP preferences in the three NHZ-terminal positions correspond to those ofthe vast majority of human leukocyte antigen class I alleles, but they represent an obstacle for peptide supply to some alleles, e.g., the B7-like group . We propose that peptides binding to these alleles, and in general, peptides with TAP affinities below a certain threshold, may be transported as extended precursors .M HC class I proteins present short breakdown products of predominantly cytosolic proteins on the cell surface to cytotoxic T lymphocytes (reviewed in reference 1) . Most of these peptides are supplied to newly assembling MHC class I proteins by the heteromeric transporter associated with antigen processing (TAP)'/TAP2' transporter complex residing in the membrane of the endoplasmic reticulum (ER) . Peptide transport by TAP can be studied using iodinated peptide substrates whose accumulation in the ER is followed grace to an integrated acceptor sequence for ER-specific glycosylation (2-4). As an alternative strategy for the study of TAP function, we recently overexpressed the human TAP complex in the insect cell/baculovirus system . In this system, peptide transport by TAP, as well as direct binding of peptide substrate to the transporter complex, can be measured (5, 6).'Abbreviations used in this paper: (3 2 m, R2-rnicroglobulin ; DTT, dithiothreitol; ER, endoplasmic reticulum ; TAP, transporter associated with antigen processing.Although the functional consequences of a polymorphism in the rat TAP2 gene clearly demonstrate that TAP selection can modify the spectrum of peptides presented by MHC class I molecules (7), it is not clear whether the human or murine transporters contribute to selection of class I-presented epitopes. Apart from a preference for peptides with a length of 8-16 residues (5,8,9), the only selectivity of the transporters known so far concerns the COOH-terminal residue. In this position, the human TAP com...

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic b-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

In early atherogenesis, subendothelial retention of lipidic droplets is associated with an inflammatory response-to-injury, culminating in the formation of foam cells and plaque. Low density lipoprotein (LDL) is the main constituent of subendothelial lipidic droplets. The process is believed to occur following LDL modification. Searching for a modified LDL in plasma, electronegative LDL [LDL(-)] was identified and found to be associated with major risk biomarkers. The apoprotein in LDL(-) is misfolded, and we show here that this modification primes the aggregation of native LDL, conforming to the typical pattern of protein amyloidogenesis. After a lag phase, whose length depends on LDL(-) concentration, light scattering and atomic force microscopy reveal early exponential growth of intermediate globules, which evolve into fibrils. These globules are remarkably similar to subendothelial droplets in atheromatous lesions and different from those produced by oxidation or biochemical manipulation. During aggregation, ellipticity and tryptophan fluorescence measurements reveal a domino-style spread of apoprotein misfolding from LDL(-) to all of the LDL. Computational analysis of the apoprotein primary sequence predicts an unstable, aggregation-prone domain in the regulatory alpha2 region. Apoprotein misfolding well represents an LDL modification able to transform this cholesterol carrier into a trigger for a response-to-injury in the artery wall.

In early phases of atherogenesis, droplets and vesicles accumulate in the subendothelial extracellular space of arterial intima. There is much evidence to suggest that these droplets, ranging between 100 and 400 nm, derive from modified low-density lipoprotein (LDL). In investigations of the formation mechanism of these droplets, LDL fusion was previously induced in vitro by proteolysis, lipolysis, oxidation, and vigorous shaking, but all treatments failed to reproduce the size distribution range of in vivo droplets, mostly resulting, instead, in particles with a diameter intermediate between that of one and two LDL. Our approach was meant to mimic LDL aging in plasma. LDL isolated from plasma that was incubated overnight at 37 degrees C is slightly modified in the secondary structure of its protein component and is primed to form very large aggregates according to a reaction-limited mechanism. This mechanism requires interactions between selected surface sites, whereas massive fusion is ruled out. In the frame of the general theory for colloids, the aggregation of LDL aged in plasma fulfills all the requirements of the reaction-limited mechanism, encompassing 1), exponential growth; 2), fractal structure, with the dimension of elementary constituent still consistent with a single LDL; and 3), extreme polydispersity of aggregates, with shape and dimension very close to that of droplets observed in vivo.

HLA-B27 molecules are interesting because of their strong association with ankylosing spondylitis (AS) and reactive arthritis (ReA). A pathogenetic role for these molecules has been postulated in presenting a putative "arthritogenic" peptide to CD8 T cells. The HLA-B*2709 subtype, although differing by a single amino acid (His116-->Asp116) from the widespread and strongly AS-associated subtype HLA-B*2705, is not found in patients. Since residue 116 interacts with the C terminus of the peptide, it is possible that the two subtypes differ in their antigen-presenting features. We show here that CD8 T cells can distinguish the two HLA-B27 subtypes when presenting a same epitope derived from Epstein-Barr virus-latent membrane protein 2. Moreover, alanine scanning mutagenesis analysis revealed that the peptide residues relevant for such recognition are different depending on whether HLA-B*2705 or -B*2709 molecules present the epitope. These results give support to the belief that functional differences determined by subtype-specific polymorphisms can have a pathogenetic relevance and open up a new scenario where subtle modifications within the peptide/HLA ligand might be responsible for the differential association between HLA-B27 subtypes and spondyloarthropathies.