Per A. Peterson

An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex

The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the αβ T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated αβ TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Vα and Vβ domains but deviates in the constant Cα domain and in the interdomain pairing of Cα with Cβ. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Cα-Cβ interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the α and β chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Vα CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vβ chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Vα and Vβ CDR3s straddle the peptide between the helices around the central position of the peptide.

Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum.

Several families of transmembrane endoplasmic reticulum (ER) proteins contain retention motifs in their cytoplasmically exposed tails. Mutational analyses demonstrated that two lysines positioned three and four or five residues from the C‐terminus represent the retention motif. The introduction of a lysine preceding the lysine that occurs three residues from the terminus of Lyt2 renders this cell surface protein a resident of the ER. Likewise, the appropriate positioning of two lysine residues in a poly‐serine sequence confines marker proteins to the ER. Arginines or histidines cannot replace lysines, suggesting that simple charge interactions are not sufficient to explain the retention. The identified consensus motif may serve as a retrieval signal that brings proteins back from a sorting compartment adjacent to the ER.

The ER-luminal domain of the HCMV glycoprotein US6 inhibits peptide translocation by TAP.

Human cytomegalovirus (HCMV) inhibits MHC class I antigen presentation by a sequential multistep process involving a family of unique short (US) region-encoded glycoproteins. US3 retains class I molecules, whereas US2 and US11 mediate the cytosolic degradation of heavy chains by the proteosomes. In US6-transfected cells, however, intracellular transport of class I molecules is impaired because of defective peptide translocation by transporters associated with antigen processing (TAP). Peptide transport is restored in HCMV mutants lacking US6. In contrast to the cytosolic herpes simplex virus protein ICP47, US6 interacts with TAP inside the endoplasmic reticulum lumen, as shown by US6 derivatives lacking the transmembrane and cytoplasmic domains and by the observation that US6 does not prevent peptides from binding to TAP. Thus, HCMV targets TAP for immune escape by a molecular mechanism different from that of herpes simplex virus.

Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum

The adenoviral transmembrane E3/19K glycoprotein is a resident of the endoplasmic reticulum. Here we show that the last six amino acid residues of the 15-membered cytoplasmic tail are necessary and sufficient for the ER retention. These residues can be transplanted onto the cytoplasmic tail of other membrane-bound proteins such that ER residency is conferred. Deletion analysis demonstrated that no single amino acid residue is responsible for the retention. The identified structural motif must occupy the extreme COOH-terminal position to be functional. An endogenous transmembrane ER protein, UDP-glucuronosyltransferase, also contains a retention signal in its cytoplasmic tail. We suggest that short linear sequences occupying the extreme COOH-terminal position of transmembrane ER proteins serve as retention signals.

The roles of iron in health and disease

Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining bodys iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.

Molecular mechanism and species specificity of tap inhibition by herpes simplex virus protein icp47

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)‐mediated translocation of antigen‐derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV‐infected cells by cytotoxic T‐lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species‐specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8‐azido‐ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP‐transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100‐fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate‐binding site of TAP.

The three-dimensional structure of retinol-binding protein.

The complex of retinol with its carrier protein, retinol‐binding protein (RBP) has been crystallized and its three‐dimensional structure determined using X‐ray crystallography. Its most striking feature is an eight‐stranded up‐and‐down beta barrel core that completely encapsulates the retinol molecule. The retinol molecule lies along the axis of the barrel with the beta‐ionone ring innermost and the tip of the isoprene tail close to the surface.

An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum.

Use of alternative initiator methionines in human invariant (Ii) chain mRNA results in the synthesis of two polypeptides, Iip33 and Iip31. After synthesis both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. Subsequently, Iip31 is transported out of the ER, guiding MHC class II to the endocytic pathway, whereas Iip33, which differs by only a 16 residue extension at the N‐terminus, becomes an ER resident. Mutagenesis of this extension showed that multiple arginines close to the N‐terminus were responsible for ER targeting. The minimal requirements of this targeting motif were found to be two arginines (RR) located at positions 2 and 3, 3 and 4 or 4 and 5 or split by a residue at positions 2 and 4 or 3 and 5. Transplanting an RR motif onto transferrin receptor demonstrated that this motif can target other type II membrane proteins to the ER. The characteristics of this RR motif are similar to the KK ER targeting motif for type I membrane proteins. Indeed, RR‐tagged transferrin receptor partially localized to the intermediate compartment, suggesting that like the KK motif, the RR motif directs the retrieval of membrane proteins to the ER via a retrograde transport pathway.

Antigen presentation and T cell development in H2-M-deficient mice.

HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.

Impaired intracellular transport of class I MHC antigens as a possible means for adenoviruses to evade immune surveillance

Early region E3 of adenovirus-2 encodes a glycoprotein, E19, that associates with class I MHC antigens. The interaction occurs concomitantly with abrogated terminal glycosylation of class I antigen heavy chains, as shown by pulse-chase and Endo H digestion experiments. Also the viral protein remains in the high mannose form throughout the chase period. These results do not reflect a general impairment of terminal glycosylation of membrane proteins. Immunofluorescence analyses demonstrated that in virally infected cells E19 and class I antigens accumulate in the perinuclear region. Class I antigens gradually disappear from the cell surface. A diminished cell surface expression of class I antigens may be a means by which adenoviruses evade immune surveillance.