S. E. Cho

Redox Regulation Facilitates Optimal Peptide Selection by MHC Class I during Antigen Processing

Activated CD8(+) T cells discriminate infected and tumor cells from normal self by recognizing MHC class I-bound peptides on the surface of antigen-presenting cells. The mechanism by which MHC class I molecules select optimal peptides against a background of prevailing suboptimal peptides and in a considerably proteolytic ER environment remained unknown. Here, we identify protein disulfide isomerase (PDI), an enzyme critical to the formation of correct disulfide bonds in proteins, as a component of the peptide-loading complex. We show that PDI stabilizes a peptide-receptive site by regulating the oxidation state of the disulfide bond in the MHC peptide-binding groove, a function that is essential for selecting optimal peptides. Furthermore, we demonstrate that human cytomegalovirus US3 protein inhibits CD8(+) T cell recognition by mediating PDI degradation, verifying the functional relevance of PDI-catalyzed peptide editing in controlling intracellular pathogens. These results establish a link between thiol-based redox regulation and antigen processing.

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation.

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2‐induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA‐mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate‐binding mutant of PDI, but not the catalytically inactive mutant, dominant‐negatively inhibited US2‐mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP‐mediated degradation of CD3δ but not Derlin‐1‐dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP‐mediated ER‐associated degradation machinery.

Human Cytomegalovirus UL18 Utilizes US6 for Evading the NK and T-Cell Responses

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses.

Receptor‐Mediated ER Export of Human MHC Class I Molecules Is Regulated by the C‐Terminal Single Amino Acid

Major histocompatibility complex class I (MHC‐I) molecules bind antigens in the endoplasmic reticulum (ER) and deliver them to the cell surface for immune surveillance of viruses and tumors. Whereas key steps of MHC‐I assembly and its acquisition of peptides in the ER are relatively well defined, little is known about how MHC‐I molecules leave the ER for cell surface expression. Here, we show that ER export of human classical MHC‐I molecules (HLA‐A/‐B/‐C) is regulated by their C‐terminal single amino acid, valine or alanine. These amino acids, conserved in nearly all known human MHC‐I alleles, serve as the ER export signal by binding to the Sec23/24 complex, a structural component of coat protein complex II (COPII) vesicles that mediate ER‐to‐Golgi trafficking. Together, our results strongly suggest that ER export of human classical MHC‐I molecules can occur via a receptor‐mediated process dictated by a highly conserved ER export signal.

Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation

Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation.

Redox-Regulated Peptide Transfer from the Transporter Associated with Antigen Processing to Major Histocompatibility Complex Class I Molecules by Protein Disulfide Isomerase

Most antigenic peptides are generated by proteasomes in the cytosol and are transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind with nascent major histocompatibilitiy complex class I molecule (MHC-I). Although the overall process of peptide-MHC-I complex assembly is well studied, the mechanism by which free peptides are delivered from TAP to MHC-I is unknown. In this study, we investigated the possible role of protein disulfide isomerase (PDI) as a peptide carrier between TAP and MHC-I. Analysis of PDI-peptide complexes reconstituted in vitro showed that PDI exhibits some degree of specificity for peptides corresponding to antigenic ligands of various human leukocyte antigen (HLA) alleles. Mutations of either anchor residues of the peptide ligand or the peptide-binding site of PDI inhibited the PDI-peptide interaction. The PDI-peptide interaction increased under reducing conditions, whereas binding of the peptide to PDI decreased under oxidizing conditions. TAP-associated PDI was predominantly present in the reduced form, whereas the MHC-I-associated PDI was present in the oxidized form. Further, upon binding of optimal peptides, PDI was released from TAP and sequentially associated with HLA-A2.1. Our data revealed a redox-regulated chaperone function of PDI in delivering antigenic peptides from TAP to MHC-I.

The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation.

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.

First report of powdery mildew caused by Erysiphe cruciferarum on chinese cabbage in China

Chinese cabbage, Brassica rapa ssp. pekinensis (syn. Brassica pekinensis (Lour.) Rupr.), in the Brassicaceae, is an important vegetable grown on about 3 million ha in China. Since 2012, a powdery mildew has been found infecting Chinese cabbage plants (cv. Qingyanchunbai No. 1) after bolting for seed production from autumn through spring 2013 in a greenhouse in Qingdao, China. Symptoms first appeared as circular to irregular white patches on both sides of the leaves, and on stems and pods, often thinly covering the whole surface. A voucher specimen was deposited in the herbarium of Qingdao Agricultural University (Accession No. HMQAU12216). Hyphae were thin-walled, smooth, hyaline, and 4 to 6 μm wide. Appressoria on the mycelia were well developed, lobed, solitary, or in pairs. Conidiophores were erect, cylindrical, 45 to 110 μm long, and comprised 3 to 4 cells. Foot-cells of conidiophores were straight, cylindrical, 16 to 28 μm long, and 7.6 to 10 μm wide. Singly-produced conidia were oblong to cylindrical or somewhat ellipsoid-doliiform, 32 to 56 × 12 to 18 μm, with a length/width ratio of 1.8 to 3.8, with angular/rectangular wrinkling of the outer wall surface, and lacked distinct fibrosin bodies. Germ tubes were produced in the perihilar position of conidia. No chasmothecia were found. These structures are typical of the powdery mildew Pseudoidium anamorph of Erysiphe (2). The specific measurements and characteristics (especially short foot-cells of conidiophores) were consistent with previous records of Erysiphe cruciferarum Opiz ex L. Junell (2,3). To confirm the identification, the complete internal transcribed spacer (ITS) region of rDNA of isolate HMQAU12216 was amplified (4) and sequenced directly. The resulting 649-bp sequence was deposited in GenBank (Accession No. KC878683). A GenBank BLAST search of ITS sequences showed an exact match with those of E. cruciferarum on B. oleracea var. acephala (GU721075) and Oidium sp. on B. pekinensis (AB522714). A pathogenicity test was conducted by gently pressing a symptomatic leaf loaded with conidia onto a leaf of each five, healthy, potted, 40-day-old plants (cv. Qingyanchunbai No. 1). Five non-inoculated plants served as a control treatment. Inoculated plants were isolated from non-inoculated plants in separate rooms in a greenhouse at 20 ± 2°C. Inoculated plants developed signs and symptoms after 10 days, whereas the control plants remained symptomless. The fungus present on the inoculated plants was identical morphologically to that originally observed on diseased plants, thus fulfilling Kochs postulates. Though many Brassica spp. have been known to be infected with E. cruciferarum throughout the world, powdery mildew of Chinese cabbage caused by E. cruciferarum has been reported only in Finland, Germany, and Korea (1,3). To our knowledge, this is the first report of powdery mildew caused by E. cruciferarum on Chinese cabbage in China. Though occurrence of the powdery mildew on Chinese cabbage was noticed in an experimental breeding plot, this finding poses a potential threat to production of this vegetable in China. References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena, Germany, 1995. (2) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, 2012. (3) H. J. Jee et al. Plant Pathol. 57:777, 2008. (4) S. Matsuda and S. Takamatsu. Mol. Phylogen. Evol. 27:314, 2003.

First report of powdery mildew of Platanus occidentalis caused by Erysiphe platani in Korea.

Platanus occidentalis L., called American sycamore or American plane, is native to North America. The trees are commonly planted throughout the world on the sides of roads and in parks. In June 2012, diseased leaves exhibiting signs of powdery mildew from a park in Daegu City of Korea were sent to Plant Clinic of Seoul National University for diagnosis. Our observations in Daegu City during September and October 2012 showed that nearly 99% of the approximately 1,000 trees surveyed were infected with a powdery mildew. Voucher specimens (n = 6) were deposited at the Korea University Herbarium (KUS). Symptoms were characterized by chlorosis, distortion, or cupping of young leaves. White superficial colonies developed amphigenously on leaves. Hyphae were flexuous to straight, branched, septate, 4 to 7 μm wide, and had lobed appressoria. Conidiophores were 120 to 350 × 5 to 7.5 μm and produced conidia singly. Foot-cells of conidiophores were straight, cylindric, and 115 to 200 μm long. Conidia were hyaline, ellipsoid-ovoid, measured 33 to 47.5 × 17.5 to 29 μm with a length/width ratio of 1.5 to 2.0, lacked distinct fibrosin bodies, and showed reticulate wrinkling of the outer walls. Germ tubes were produced on the subterminal position of conidia. No chasmothecia were observed. The structures and measurements were compatible with those of the anamorphic state of Erysiphe platani (Howe) U. Braun & S. Takam. (1). To confirm the identification, the complete internal transcribed spacer (ITS) region of the rDNA from isolate KUS-F26959 was amplified with nested PCR and sequenced. The resulting sequence of 625 bp was deposited in GenBank (Accession No. JX997805). A GenBank BLAST search of this sequence showed only one base substitution with the four sequences (JQ365940 to JQ365943) of E. platani on Platanus spp. Pathogenicity was confirmed through inoculation tests by gently pressing diseased leaves onto young leaves of three 2-year-old disease-free seedlings. Three non-inoculated plants were used as control. Plants were maintained in a greenhouse at 24 to 30°C. Inoculated leaves developed symptoms after 7 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the original diseased leaves, fulfilling Kochs postulates. Since E. platani first was recorded in the United States in 1874, it has been regarded as endemic in North America. From the second half of the 20th century, introduction and expansion of the range of this fungus to South America, South Africa, Australia and New Zealand, Europe, and Asia have been reported (1,2). To our knowledge, this is the first report of E. platani infections of P. occidentalis in Korea. This species was recorded on P.× hispanica from Japan in 1999 (4) and on P. orientalis from China in 2006 (3), suggesting invasive spread of the sycamore powdery mildew in East Asia. Since American sycamores are widely planted in Korea, control measures should be made to prevent further spread of the disease. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No.11. CBS, Utrecht, 2012. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved October 22, 2012. (3) C. Liang et al. Plant Pathol. 57:375, 2008. (4) S, Tanda. J. Agric. Sci., Tokyo Univ. Agric. 43:253, 1999.