Playing hide and seek: Tumor cells in control of MHC class I antigen presentation.

MHC class I (MHC-I) molecules present a blueprint of the intracellular proteome to T cells allowing them to control infection or malignant transformation. As a response, pathogens and tumor cells often downmodulate MHC-I mediated antigen presentation to escape from immune surveillance. Although the fundamental rules of antigen presentation are known in detail, the players in this system are not saturated and new modules of regulation have recently been uncovered. Here, we update the understanding of antigen presentation by MHC-I molecules and how this can be exploited by tumors to prevent exposure of the intracellular proteome. This knowledge can provide new ways to improve immune responses against tumors and pathogens.

PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance.

Protein kinase A (PKA)-induced estrogen receptor alpha (ERα) phosphorylation at serine residue 305 (ERαS305-P) can induce tamoxifen (TAM) resistance in breast cancer. How this phospho-modification affects ERα specificity and translates into TAM resistance is unclear. Here, we show that S305-P modification of ERα reprograms the receptor, redirecting it to new transcriptional start sites, thus modulating the transcriptome. By altering the chromatin-binding pattern, Ser305 phosphorylation of ERα translates into a 26-gene expression classifier that identifies breast cancer patients with a poor disease outcome after TAM treatment. MYC-target genes and networks were significantly enriched in this gene classifier that includes a number of selective targets for ERαS305-P. The enhanced expression of MYC increased cell proliferation in the presence of TAM. We demonstrate that activation of the PKA signaling pathway alters the transcriptome by redirecting ERα to new transcriptional start sites, resulting in altered transcription and TAM resistance.

Phosphorylation of the oestrogen receptor alpha at serine 305 and prediction of tamoxifen resistance in breast cancer.

Phosphorylation of oestrogen receptor alpha at serine 305 (ERalphaS305-P) induces tamoxifen resistance in experimental studies, but does not influence response to other endocrine agents, such as fulvestrant. We evaluated ERalphaS305-P using immunohistochemistry in 377 breast carcinomas from premenopausal participants of a randomized trial (n=248) and patients with advanced disease (n=129). Among the premenopausal patients, adjuvant tamoxifen improved recurrence-free survival (RFS) for ERalphaS305-P-negative tumours (multivariate HR=0.53, 95% CI 0.32-0.86, p=0.010), but not for ERalphaS305-P-positive tumours (multivariate HR=1.01, 95% CI 0.33-3.05, p=0.99) (interaction p=0.131). Notably, ERalphaS305-P was not significantly associated with RFS in patients not treated with tamoxifen (multivariate HR=0.64, 95% CI 0.30-1.37, p=0.248), indicating that ERalphaS305-P is a marker for treatment outcome rather than tumour progression. Given the direct experimental link between ERalphaS305-P and tamoxifen resistance and these first clinical data suggesting that premenopausal patients with ERalphaS305-P-positive breast cancer are resistant to adjuvant tamoxifen, further research is encouraged to study whether alternative endocrine treatment should be considered for this subgroup.

The ins and outs of intracellular peptides and antigen presentation by MHC class I molecules.

MHC class I molecules present small intracellular generated fragments to the outside surveying immune system. This is the result of a series of biochemical processes involving biosynthesis, degradation, translocation, intracellular transport, diffusion, and many more. Critical intermediates and end products of this cascade of events are peptides. The peptides are generated by the proteasome, degraded by peptidases unless transported into the ER where another peptidase and MHC class I molecules are waiting. Unless peptides bind to MHC class I molecules, they are released from the ER and enter the cytoplasm by a system resembling the ERAD pathway in many aspects. The cycle of peptides over the ER membrane with the proteasome at the input site and peptidases or MHC class I molecules on the output site are central in the MHC class I antigen presentation pathway and this review.

[The 2004 Nobel Prize in Chemistry for the discovery of ubiquitin-mediated protein degradation]. / Nobelprijs Scheikunde 2004 voor de ontdekking van ubiquitine-gemedieerde eiwitafbraak.

This year's Nobel Prize in Chemistry has been awarded to Aaron Ciechanover, Avram Herskho and Irwin Rose for the discovery of ubiquitin-mediated protein degradation. In a series of groundbreaking experiments these scientists described the basic principles for a unique posttranslational modification based on the conjugation of the small protein ubiquitin to proteins deemed for degradation. Although ubiquitin started in 1980 as an unusual modification of certain proteins, it is now clear that it functions as a signal for degradation when it forms a polymer. Hundreds of proteins are involved in the controlled destruction of ubiquitin-labelled proteins in the cell. And hundreds of other proteins are involved in protein modification by mono-ubiquitin, so that other processes, such as the formation of another degradation compartment, the lysosome, can proceed normally.

Bovine herpesvirus 1 interferes with TAP-dependent peptide transport and intracellular trafficking of MHC class I molecules in human cells.

Bovine herpesvirus 1 (BoHV-1), the cause of infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle, establishes a lifelong infection, despite the presence of antiviral immunity in the host. BoHV-1 has been shown to elude the host immune system, but the viral gene products responsible for this interference have not yet been identified. Studies aiming at the identification of BoHV-1-encoded immune evasion genes have been hampered by the lack of bovine-specific immunological reagents. Some of the immune evasion molecules identified for other herpesviruses are host species specific; others can act across the species barrier. In this study, experiments were performed to investigate whether BoHV-1 can infect human cells and interfere with antigen processing and presentation in these cells. A human melanoma cell line, Mel JuSo, appeared to be permissive for BoHV-1 infection. BoHV-1 induced expression of major viral glycoproteins at the surface of these cells and produced progeny virus up to 10(5) plaque forming units per ml. BoHV-1 infection resulted in impaired intracellular transport of human MHC class I molecules and inhibition of human TAP. These data indicate that the BoHV-1-encoded molecule(s) that block antigen presentation in bovine cells are able to interact with homologous components of the human MHC class I presentation pathway. The fact that immune evasion by BoHV-1 can be studied in human cells will facilitate the identification of the BoHV-1 gene products involved in this process. Moreover, the data presented here suggest that the BoHV-1 encoded inhibitors of antigen presentation represent potential immune suppressive agents for use in humans.

[The immunological synapse: an exclusive zone for directed cell death after specific recognition]. / De immunologische synaps: een exclusieve zone voor gerichte celdood na specifieke herkenning.

Cytotoxic T lymphocytes or killer cells are an important part of the immune system. These cells move through the body while their T cell receptors initiate low affinity contacts with MHC class I proteins (also termed transplantation antigens) on other cells. If there is a specific interaction between the T cell receptor and MHC class I molecule, then that cell is killed. This happens with virus-infected cells but also with tumour cells, during transplant rejection and in various autoimmune diseases. The immunological synapse is a sealed area generated by the killer cell in order to selectively kill a particular cell. The cytotoxic content of the killer cell is released into this area.

Herpes viral proteins manipulating the peptide transporter TAP.

The peptide transporter associated with antigen processing (TAP) is crucial for class I-restricted antigen presentation because it transfers cytosolic peptides into the endoplasmic reticulum (ER) lumen for class I binding. It is therefore not surprising that TAP is targeted for inactivation by many viruses. Herpesviruses have been very successful in designing various proteins that inactivate TAP. We summarise current knowledge on the class I antigen presentation pathway and the function, structure and action of TAP and its viral inhibitors.

Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance.

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.

In vivo and in vitro modulation of HLA-DM and HLA-DO is induced by B lymphocyte activation.

Ag presentation via HLA class II molecules in B lymphocytes depends on the coordinated action of HLA-DM, the catalyst of class II-peptide loading, and HLA-DO, a pH-dependent modulator of DM, the expression of which is almost completely restricted to B lymphocytes. The relative expression levels of both class II modulators are critical for the composition of the HLA class II peptide repertoire. The data in this work demonstrate that DO and DM expression are both dependent on the cellular activation status in primary human B lymphocytes. In vivo low-density activated primary human B lymphocytes show a prominent reduction in DO and DM expression when compared with high-density resting primary B lymphocytes. In vitro, reduction of DO and DM expression can be induced by B lymphocyte activation via the B cell receptor or by use of the phorbol ester, PMA. Specific inhibition of protein kinase C resulted in a significant reduction of HLA-DO and is potentially due to protein degradation in lysosomal compartments as the phenomenon is reversed by chloroquine. Thus, the expression of the dedicated HLA class II chaperone DM and its pH-dependent modulator DO is regulated and tightly controlled by the activation status of the B lymphocyte.

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body.

MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.

The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide.

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.

From fixed to FRAP: measuring protein mobility and activity in living cells.

Experiments with fluorescence recovery after photobleaching (FRAP) started 30 years ago to visualize the lateral mobility and dynamics of fluorescent proteins in living cells. Its popularity increased when non-invasive fluorescent tagging became possible with the green fluorescent protein (GFP). Many researchers use GFP to study the localization of fusion proteins in fixed or living cells, but the same fluorescent proteins can also be used to study protein mobility in living cells. Here we review the potential of FRAP to study protein dynamics and activity within a single living cell. These measurements can be made with most standard confocal laser-scanning microscopes equipped with photobleaching protocols.

Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells.

Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.

Determining protein transport to the plasma membrane.

Many integral membrane proteins synthesized in the endoplasmic reticulum ultimately arrive at the cell surface to contact the cell environment. During transit through the Golgi and trans-Golgi network, proteins acquire post-translational modifications that can be used to track the appearance of such modified proteins at the cell surface. Cellular proteins can be treated with enzymes-e.g., sialidase or protease-or antibodies, or biotinylated to identify molecules that have reached the cell surface. Some proteins first enter the endocytic pathway before appearing at the cell surface; this is detected by treating the cells at 4 degrees and 37 degrees C. Analysis of the number of sialic acids on proteins of cells treated at 4 degrees C identifies proteins resident at the cell surface, while cells treated at 37 degrees C internalize the sialidase, which can then act with proteins in the endocytic compartments.

What to do with HLA-DO?

Antigenic peptide binding to MHC class II molecules in the endocytic pathway occurs via a multifactorial process that requires the support of a specialized lysosomal chaperone called HLA-DM. DM shows both in primary amino acid sequence and quaternary structure a high homology to both MHC class I and class II molecules. Like the peptide presenting class II molecules, DM is expressed in all professional antigen presenting cells. DM catalyzes the dissociation of peptides that do not bind stably to the class II peptide-binding groove, thereby leading to the preferential presentation of stably binding antigenic peptides. The recently discovered HLA-DO molecule is mainly expressed in B cells and associates with DM, thereby markedly affecting DM function. Like DM, the genes encoding the HLA-DO heterodimer lie within the MHC class II region and exhibit strong homology to classical class II molecules. This review evaluates the unique effects of DO on DM-mediated antigen presentation by MHC class II molecules and discusses the possible physiological relevance for the B cell-specific expression of DO and its function.

Export of antigenic peptides from the endoplasmic reticulum intersects with retrograde protein translocation through the Sec61p channel.

Antigenic peptides are translocated by the TAP peptide transporter from the cytosol into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. Peptides that fail to bind need to be removed from the ER. Here we provide evidence that peptide export utilizes the Sec61p translocon as demonstrated by blocking this channel with bacterial exotoxin. Peptide export interferes with the retrotranslocation of beta2-microglobulin from the ER to the cytosol, suggesting similar pathways for the disposal of proteins and oligopeptides. Peptide export requires ATP supply to the ER lumen but is independent of ATP hydrolysis.