[Show abstract][Hide abstract] ABSTRACT: MHC class I molecules present peptides derived from intracellular proteins, enabling immune surveillance by CD8(+) T cells and the elimination of virus-infected and cancerous cells. It has been argued that the dominant source of MHC class I-presented peptides is through proteasomal degradation of newly synthesized defective proteins, termed defective ribosomal products (DRiPs). Here, we critically examine the DRiP hypothesis and discuss recent studies indicating that antigenic peptides are generated from the entire proteome and not just from failures in protein synthesis or folding.
Trends in Immunology 02/2014; · 9.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When cells die by necrosis in vivo they stimulate an inflammatory response. It is thought that this response is triggered when the injured cells expose proinflammatory molecules, collectively referred to as damage associated molecular patterns (DAMPs), which are recognized by cells or soluble molecules of the innate or adaptive immune system. Several putative DAMPs and/or their receptors have been identified, but whether and how much they participate in responses in vivo is incompletely understood, and they have not previously been compared side-by-side in the same models. This study focuses on evaluating the contribution of multiple mechanisms that have been proposed to or potentially could participate in cell death-induced inflammation: The third component of complement (C3), ATP (and its receptor P2X7), antibodies, the C-type lectin receptor Mincle (Clec4e), and protease-activated receptor 2 (PAR2). We investigate the role of these factors in cell death-induced inflammation to dead cells in the peritoneum and acetaminophen-induced liver damage. We find that mice deficient in antibody, C3 or PAR2 have impaired inflammatory responses to dying cells. In contrast there was no reduction in inflammation to cell death in the peritoneum or liver of mice that genetically lack Mincle, the P2X7 receptor or that were treated with apyrase to deplete ATP. These results indicate that antibody, complement and PAR2 contribute to cell death-induced inflammation but that Mincle and ATP- P2X7 receptor are not required for this response in at least 2 different in vivo models.
PLoS ONE 01/2014; 9(8):e104741. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin of the MHC class I-presented peptides are thought to be primarily from newly synthesized but defective proteins, termed defective ribosomal products. Most of the data supporting this concept come from studies in which inhibitors of protein synthesis were found to rapidly block Ag presentation even when cells contained a pool of mature proteins. However, these data only indirectly address the origin of presented peptides, and in most studies, the contribution of mature functional proteins to the class I peptide pool has not been directly quantified. In this report, we address the efficiency and contribution of mature proteins using a tetracycline-inducible system to express Ags that are conditionally stabilized upon ligand binding. This system circumvents the use of general inhibitors of protein synthesis to control Ag expression. Moreover, by controlling Ag stabilization, we could investigate whether the degradation of mature Ags contributed to Ag presentation at early and/or late time points. We show that mature proteins are the major contributor of peptides presented on class I for two distinct antigenic constructs. Furthermore, our data show that the protein synthesis inhibitors used previously to test the contribution of defective proteins actually block Ag presentation in ways that are independent from blocking Ag synthesis. These data suggest that for the constructs we have analyzed, mature functional proteins, rather than defective ribosomal products, are the predominant source of MHC class I-presented peptides.
The Journal of Immunology 10/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study, we have found that intestinal flora strongly influence peritoneal neutrophilic inflammatory responses to diverse stimuli including pathogen-derived particles like zymosan and sterile irritant particles like crystals. When germ-free and flora-deficient (antibiotic-treated) mice are challenged with zymosan intraperitoneally, neutrophils are markedly impaired in their ability to extravasate from blood into the peritoneum. In contrast, in these animals, neutrophils can extravasate in response to an intraperitoneal injection of the chemokine, macrophage inflammatory protein 2 (MIP-2). Neutrophil recruitment upon inflammatory challenge requires stimulation by microbiota through a myeloid differentiation primary response gene (88) (MyD88)-dependent pathway. MyD88 signaling is crucial during the development of the immune system but depending upon the ligand it may be dispensable at the time of the actual inflammatory challenge. Furthermore, pre-treatment of flora-deficient mice with a purified MyD88-pathway agonist is sufficient to restore neutrophil migration. In summary, this study provides insight into the role of gut microbiota in influencing acute inflammation at sites outside of the gastrointestinal tract. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: All vertebrate nucleated cells generate peptides from their expressed gene products and then display them at the cell surface bound to MHC class I molecules. This allows CD8(+) T cells to detect and eliminate abnormal cells that are synthesizing foreign proteins, e.g., from viruses or mutations. To permit the immune system to more uniformly monitor a cell's proteins, regardless of their half-life or location, it has been thought that the products of rapid degradation of the mistakes of protein synthesis (defective ribosomal products, DRiPs) preferentially contribute to the class I-presented peptides. However, using intein catalysis to generate peptide sequences exclusively by posttranslational splicing of mature proteins, we show here that presented peptides can be generated from fully folded and functional proteins. Remarkably, the presentation of peptides from two model mature proteins is just as efficient as from newly synthesized proteins subject to errors in translation or folding. These results indicate that for the constructs we have analyzed, DRiPs are not a more efficient source of class I peptides for antigen presentation than the turnover of mature functional proteins. Accordingly, our data suggest that one of the major ways the immune system evaluates the health of cells is by monitoring the breakdown products of the proteome.
Proceedings of the National Academy of Sciences 10/2012; 109(42):16998-7003. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Uric acid is a waste product of purine catabolism. This molecule comes to clinical attention when it nucleates to form crystals of monosodium urate (MSU) in joints or other tissues, and thereby causes the inflammatory disease of gout. Patients with gout frequently suffer from a number of comorbid conditions including hypertension, diabetes mellitus and cardiovascular disease. Why MSU crystals trigger inflammation and are associated with comorbidities of gout has been unclear, but recent studies provide new insights into these issues. Rather than simply being a waste product, uric acid could serve a pathophysiological role as a local alarm signal that alerts the immune system to cell injury and helps to trigger both innate and adaptive immune responses. The inflammatory component of these immune responses is caused when urate crystals trigger both inflammasome-dependent and independent pathways to generate the proinflammatory cytokine IL-1. The resulting bioactive IL-1 stimulates the inflammation of gout and might contribute to the development of other comorbidities. Surprisingly, the same mechanisms underlie the inflammatory response to a number of irritant particles, many of which also cause disease. These new insights help to explain the pathogenesis of gout and point to potential new therapeutic targets for this and other sterile inflammatory diseases.
[Show abstract][Hide abstract] ABSTRACT: The sterile inflammatory response to cell death and irritant crystals is medically important because it causes disease. Although these stimuli are structurally distinct, they cause inflammation through a common pathway that requires the cytokine IL-1. In vitro, the inflammasome, and in particular its generation of active caspase-1, is absolutely required to produce bioactive IL-1β. However, in this study, we report that caspase-1 is not required in vivo for much of the IL-1β-dependent sterile inflammatory response. Furthermore, we find that cathepsin C, which controls the activity of a number of leukocyte serine proteases capable of processing IL-1β, plays a major role in this caspase-1-independent pathway. Mice that are deficient in cathepsin C have reduced inflammatory responses to dying cells and silica crystals. In the absence of cathepsin C, caspase-1 becomes rate limiting such that mice doubly deficient in both of these proteases make little IL-1β in vivo and have markedly attenuated inflammatory responses to the sterile stimuli. In contrast, these mutant mice generate normal inflammation in response to exogenous IL-1β, indicating that cathepsin C and caspase-1 function upstream of IL-1β, and, in their absence, all components of the pathway downstream of mature IL-1β are intact.
The Journal of Immunology 08/2012; 189(7):3734-40. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein homeostasis depends on a balance of translation, folding, and degradation. Here, we demonstrate that mild inhibition of translation results in a dramatic and disproportional reduction in production of misfolded polypeptides in mammalian cells, suggesting an improved folding of newly synthesized proteins. Indeed, inhibition of translation elongation, which slightly attenuated levels of a copepod GFP mutant protein, significantly enhanced its function. In contrast, inhibition of translation initiation had minimal effects on copepod GFP folding. On the other hand, mild suppression of either translation elongation or initiation corrected folding defects of the disease-associated cystic fibrosis transmembrane conductance regulator mutant F508del. We propose that modulation of translation can be used as a novel approach to improve overall proteostasis in mammalian cells, as well as functions of disease-associated mutant proteins with folding deficiencies.
Journal of Biological Chemistry 08/2012; 287(41):34264-72. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Radiation injury in the skin causes radiodermatitis, a condition in which the skin becomes inflamed and the epidermis can break down. This condition causes significant morbidity and if severe it can be an independent factor that contributes to radiation mortality. Radiodermatitis is seen in some settings of radiotherapy for cancer and is also of concern as a complication post-radiation exposure from accidents or weapons, such as a "dirty bomb". The pathogenesis of this condition is incompletely understood. Here we have developed a murine model of radiodermatitis wherein the skin is selectively injured by irradiation with high-energy electrons. Using this model we showed that the interleukin-1 (IL-1) pathway plays a significant role in the development of radiodermatitis. Mice that lack either IL-1 or the IL-1 receptor developed less inflammation and less severe pathological changes in their skin, especially at later time-points. These findings suggest that IL-1 pathway may be a potential therapeutic target for reducing the severity of radiodermatitis.
Radiation Research 08/2012; 178(3):166-72. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A r t i c l e s Proteasomes are vital in generating peptides for presentation on MHC class I molecules 1 . Each proteasome consists of 14 structural subunits and 6 catalytic subunits (two each of the β1, β2 and β5 subunits) 2 . In addition to these three catalytic subunits, three alternative catalytic subunits denoted β1i (LMP2 or Psmb9), β2i (MECL1 or Psmb10) and β5i (LMP7 or Psmb8) are constitutively expressed in a number of hematopoietic cells and are induced in other cell types by interferon-γ (IFN-γ) 2,3 . When expressed, these alternative subunits are preferen-tially incorporated into newly assembling complexes to form immuno-proteasomes 3 and change the catalytic activities of these complexes. Compared with the constitutive proteasomes, immunoproteasomes cleave more rapidly after hydrophobic and basic amino acid residues and less rapidly after acidic ones 4–6 . As peptides with hydrophobic or sometimes basic C termini preferentially bind to MHC class I mol-ecules 7 , it has long been suggested that immunoproteasomes have a specialized role in creating antigenic peptides. However, mice lacking individual immunoproteasome catalytic subunits have relatively modest changes in antigen presentation. For example, β5i-deficient mice have moderately (~50%) lower MHC class I surface expression 8 and lower or higher efficiency in presenting only a few epitopes, whereas the majority of immunogenic peptides examined are pre-sented normally 8–15 . The published analyses have examined only the presentation of known epitopes, and it is unknown whether and how often immunoproteasome-deficient mice present different peptides compared with wild-type mice. To determine whether the modest changes in these mice were due to some contribution from the remaining immunoproteasome catalytic subunits, we created a mouse that was triply deficient, lacking all three immune subunits. Because the genes encoding β1i (Psmb9) and β5i (Psmb8) are so close together on chromosome 17, which made the chance of generating a doubly deficient mouse by crossing β5i-null with β1i-null mice vanishingly small, we chose to create a new sequential deletion of these two genes. We then bred the β1i and β5i doubly deficient mice to β2i-deficient (Psmb10-null) mice to create the animal with a triple immunoproteasome deficiency. We found that the triply deficient mice had altered presentation of most of the epitopes we tested, both in vitro and in vivo, and that these changes in antigen presentation were sufficient to cause triply deficient mice to reject wild-type cells. RESULTS Generation of immunoproteasome–triply deficient mice To generate the β1i β5i doubly deficient animals, we designed a sequential deletion strategy (Fig. 1a). First a LacZ-FRT-neo-FRT construct was fused in frame to the start codon (27 base pairs (bp) downstream of the 5′ end of exon 1) of Psmb8 (which encodes β5i), removing the remainder of exon 1 plus exons 2 through 5 by homolo-gous recombination. The neo gene was then removed by enhanced FLP recombinase (FLPe) activity in the bacteria. An alkaline phosphatase–loxP-neo-loxP construct was then fused in frame to the
[Show abstract][Hide abstract] ABSTRACT: The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes had not been available. Having now developed mice lacking the three immunoproteasome catalytic subunits, we found that the dendritic cells of these mice had defects in presenting several major histocompatibility complex (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes was markedly reduced in immunoproteasome-deficient animals compared with wild-type animals, whereas presentation of MHC class II peptides was unaffected. According to mass spectrometry, the repertoire of MHC class I-presented peptides was ∼50% different from that in wild-type mice, and these differences were sufficient to stimulate robust transplant rejection of wild-type cells in mutant mice. These results indicated that immunoproteasomes were more important in antigen presentation than previously thought.
[Show abstract][Hide abstract] ABSTRACT: The expression of IMP3, an oncofetal protein, has been strongly associated with aggressive cancers. In this study, we investigated whether IMP3 can serve as a biomarker to predict invasive squamous cell carcinoma (SCC) in patients with cervical intraepithelial neoplasia (CIN) II and III. A total of 1249 patients with no dysplasia, CINs, or invasive SCC were studied for IMP3 expression. The 710 patients with CIN II and III in their cervical biopsies were further evaluated for invasive cancer-free survival analysis. The role of IMP3 in the regulation of cell proliferation and migration of HeLa cervical cancer cells was examined by modification of IMP3 expression with small interference RNA. Compared with CIN I or cervical tissues without dysplasia, IMP3 expression was significantly increased not only in invasive SCC but also most importantly in a subset of CIN III cases with concurrent invasive SCC. Importantly, invasive cancer was found only in patients with IMP3-positive CIN II and III, whereas no invasive cancer was detected in patients with IMP3-negative CIN II and III in their follow-up resections (P<0.0001). Reduction of IMP3 expression in cervical cancer cells significantly reduced cell migration without altering cell proliferation. IMP3 plays a critical role in the development of invasive SCC from cervical dysplasia. IMP3 can be used at the time of initial diagnosis of CIN to identify a group of patients with an increased chance of developing invasive cancer.
The American journal of surgical pathology 11/2011; 35(11):1638-45. · 4.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immune system plays an essential role in protecting the host against infections and to accomplish this task has evolved mechanisms to recognize microbes and destroy them. In addition, it monitors the health of cells and responds to ones that have been injured and killed, even if this occurs under sterile conditions. This process is initiated when dying cells expose intracellular molecules that can be recognized by cells of the innate immune system. As a consequence of this recognition, dendritic cells are activated in ways that help to promote T-cell responses to antigens associated with the dying cells. In addition, macrophages are stimulated to produce the cytokine interleukin-1 that then acts on radioresistant parenchymal cells in the host in ways that drive a robust inflammatory response. In addition to dead cells, a number of other sterile particles and altered physiological states can similarly stimulate an inflammatory response and do so through common pathways involving the inflammasome and interleukin-1. These pathways underlie the pathogenesis of a number of diseases.
[Show abstract][Hide abstract] ABSTRACT: One of the major clinical challenges is to predict recurrence of meningioma. Recently, we have found that IMP3, an oncofetal RNA-binding protein, is a biomarker to predict aggressive tumors.
To investigate whether IMP3 can be used as a new biomarker to predict the recurrence and overall survival of patients with meningiomas.
One hundred seven patients with primary meningiomas were investigated for expression of IMP3 by immunohistochemistry and whether expression of this molecule correlated with tumor recurrence and overall survival.
Tumor recurrence was found in 13 of 107 patients with primary meningioma. Seven of 107 patients (6.5%) with primary meningiomas expressed IMP3. Kaplan-Meier plots and log-rank tests showed that patients with IMP3-positive tumors had a much higher recurrence rate (P = .004) and a poorer overall survival (P < .001) than did patients with IMP3-negative tumors. The 5-year recurrence-free and overall survival rates were 0% and 36% in IMP3-positive patients versus 89% and 94% in IMP3-negative patients, respectively. Multivariable analysis of IMP3 status (positive versus negative) in primary tumors showed a hazard ratio of 17.89 for recurrence (P = .01), which was much higher than hazard ratios associated with all other known risk factors including higher tumor grades and Ki-67 labeling index.
IMP3 is a potential independent prognostic biomarker that can be used at the time of initial diagnosis of meningioma to identify patients who have a high risk of developing a recurrence.
Archives of pathology & laboratory medicine 08/2011; 135(8):1032-6. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The distinction of malignant mesothelioma from reactive mesothelial proliferation remains to be a major challenge for surgical pathologists. In this study, we investigated whether insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3), an oncofetal protein, can be used as a biomarker to distinguish between malignant and reactive mesothelial cells. A total of 109 cases (mesothelioma, n=45; reactive mesothelial proliferation, n=64) were examined by immunohistochemistry for IMP3 expression. IMP3 showed strong cytoplasmic staining in 33 of 45 (73%) mesothelioma cases. In contrast, the expression of IMP3 was undetectable in all (64 cases) benign reactive mesothelial proliferations. Among the IMP3-positive mesotheliomas, 27 (82%) exhibited diffuse IMP3 expression. The vast majority of IMP3-positive subtypes of mesotheliomas showed IMP3 expression in >50% of malignant cells, as this diffuse staining pattern occurred in 17 (81%) cases of epithelial, 4 (100%) cases of sarcomatoid, and 6 (75%) cases of mixed types of mesothelioma. In addition, 2 cases, which were initially diagnosed as atypical mesothelial proliferations and later confirmed to be mesotheliomas, showed diffuse IMP3 expression. Our findings suggest that IMP3 is a new positive biomarker for malignant mesothelioma. IMP3 immunohistochemical staining can be used as an adjunct tool in the distinction of malignant mesothelioma from reactive mesothelial proliferations.
The American journal of surgical pathology 06/2011; 35(6):878-82. · 4.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.
[Show abstract][Hide abstract] ABSTRACT: A major function of proteasomes and macroautophagy is to eliminate misfolded potentially toxic proteins. Mammalian proteasomes, however, cannot cleave polyglutamine (polyQ) sequences and seem to release polyQ-rich peptides. Puromycin-sensitive aminopeptidase (PSA) is the only cytosolic enzyme able to digest polyQ sequences. We tested whether PSA can protect against accumulation of polyQ fragments. In cultured cells, Drosophila and mouse muscles, PSA inhibition or knockdown increased aggregate content and toxicity of polyQ-expanded huntingtin exon 1. Conversely, PSA overexpression decreased aggregate content and toxicity. PSA inhibition also increased the levels of polyQ-expanded ataxin-3 as well as mutant α-synuclein and superoxide dismutase 1. These protective effects result from an unexpected ability of PSA to enhance macroautophagy. PSA overexpression increased, and PSA knockdown or inhibition reduced microtubule-associated protein 1 light chain 3-II (LC3-II) levels and the amount of protein degradation sensitive to inhibitors of lysosomal function and autophagy. Thus, by promoting autophagic protein clearance, PSA helps protect against accumulation of aggregation-prone proteins and proteotoxicity.
Human Molecular Genetics 12/2010; 19(23):4573-86. · 7.69 Impact Factor