Tomo Saric

An IFN-γ–induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I–presented peptides

Precursors to major histocompatibility complex (MHC) class I–presented peptides with extra NH2-terminal residues can be efficiently trimmed to mature epitopes in the endoplasmic reticulum (ER). Here, we purified from liver microsomes a lumenal, soluble aminopeptidase that removes NH2-terminal residues from many antigenic precursors. It was identified as a metallopeptidase named “adipocyte-derived leucine” or “puromycin-insensitive leucine-specific” aminopeptidase. However, because we localized it to the ER, we propose it be renamed ER–aminopeptidase 1 (ERAP1). ERAP1 is inhibited by agents that block precursor trimming in ER vesicles and although it trimmed NH2-extended precursors, it spared presented peptides of 8 amino acid and less. Like other proteins involved in antigen presentation, ERAP1 is induced by interferon-γ. When overexpressed in vivo, we found that ERAP1 stimulates the processing and presentation of an antigenic precursor in the ER.

The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8–9 residues

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) appears to be specialized to produce peptides presented on class I major histocompatibility complex molecules. We found that purified ERAP1 trimmed peptides that were ten residues or longer, but spared eight-residue peptides. In vivo, ERAP1 enhanced production of an eight-residue ovalbumin epitope from precursors extended on the NH2 terminus that were generated either in the ER or cytosol. Purified ERAP1 also trimmed nearly half the nine-residue peptides tested. By destroying such nine-residue peptides in normal human cells, ERAP1 reduced the overall supply of antigenic peptides. However, after interferon-γ treatment, which causes proteasomes to produce more NH2-extended antigenic precursors, ERAP1 increased the supply of peptides for MHC class I antigen presentation.

The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides

Three different proteolytic processes have been shown to be important in the generation of antigenic peptides displayed on MHC-class I molecules. The great majority of these peoptides are derived from oligopeptides produced during the degradation of intracellular proteins by the ubiquitin-proteasome pathway. Novel methods were developed to follow this process in vitro. When pure 26S proteasomes degrade the model substrate, ovalbumin, they produce the immunodominant peptide, SIINFEKL, occasionally, but more often an N-extended form of SIINFEKL. Interferon-gamma stimulates antigen presentation in part by inducing new forms of the proteasome that are more efficient in antigen presentation, and in vitro these immunoproteasomes specifically produce more of the N-extended versions of SIINFEKL. In addition, gamma-interferon induces a novel 26S complex containing the 19S and 20S particles and the proteasome activator, PA28, which we show cleaves proteins in distinct ways. In vivo studies established that proteasomal cleavages produce the C-termini of antigenic peptides, but not their N-termini, which can be formed efficiently by aminopeptidases that trim longer proteasomal products to the presented epitopes. gamma-interferon stimulates this trimming process by inducing in the cytosol leucine aminopeptidase and a novel aminopeptidase in the ER. Peptides released by proteasomes, including antigenic peptides, are labile in cytosolic extracts, and most of the longer proteasome products are rapidly cleaved by the cytosolic enzyme, thymet oligopeptidase (TOP). If cells express large amounts of TOP, class I presentation decreases, and if TOP is inhibited, presentation increases. Thus, peptide degradation in the cytosol appears to limit the efficiency of antigen presentation.

Protein degradation and the generation of MHC class I-presented peptides

Over the past decade there has been considerable progress in understanding how MHC class I-presented peptides are generated. The emerging theme is that the immune system has not evolved its own specialized proteolytic mechanisms but instead utilizes the phylogenetically ancient catabolic pathways that continually turnover proteins in all cells. Three distinct proteolytic steps have now been defined in MHC class I antigen presentation. The first step is the degradation of proteins by the ubiquitin-proteasome pathway into oligopeptides that either are of the correct size for presentation or are extended on their amino-termini. In the second step, aminopeptidases trim N-extended precursors into peptides of the correct length to be presented on class I molecules. The third step involves the destruction of peptides by endo- and exopeptidases, which limits antigen presentation, but is important for preventing the accumulation of peptides and recycling them back to amino acids for protein synthesis or production of energy. The immune system has evolved several components that modify the activity of these ancient pathways in ways that enhance the generation of class I-presented peptides. These include catalytically active subunits of the proteasome, the PA28 proteasome activator, and leucine aminopeptidase, all of which are upregulated by interferon-gamma. In addition to these pathways that operate in all cells, dendritic cells and macrophages can also generate class I-presented peptides from proteins internalized from the extracellular fluids by degrading them in endocytic compartments or transferring them to the cyotosol for degradation by proteasomes.

In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c‐Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS‐cell‐based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RTPCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, αMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac‐specific voltage‐gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels;CMs generated from iPS cells have a ventricular phenotype;and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC‐derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.—Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., Saric, T., Wernig, M., Jaenisch, R., Hescheler, J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23, 4168‐4180 (2009). www.fasebj.org

Indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection

Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC‐CMs) and iPS cells (iPS‐CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5–7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force‐frequency relationship (FFR), effects of Ca2+, Ni2+, nifedipine, ryanodine, β‐adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC‐CMs and iPS‐CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca2+ and decreased with nifedipine. Force was reduced by Ni2+. Isoproterenol (1 µM) increased the force most pronounced in embryonic tissue (207±31%, n=7;ESC‐CMs: 123±5%, n=4; iPS‐CMs: 120 ±4%, n=8). EC50 values were similar. Contractile properties of iPS‐CMs and ESC‐CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the β‐adrenergic response of iPS‐CMs and ESC‐CMs.—Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Šarić, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J. 24, 2739–2751 (2010). www.fasebj.org

Genetic pattern of prostate cancer progression

Genetic alterations in primary prostate cancer (CaP) have been extensively studied, yet little is known about the genetic mechanisms underlying progression of primary CaP to metastatic prostate cancer. As a result, it is not possible to distinguish clinically indolent localized disease from potentially life‐threatening tumors with high metastatic potential. To address this question, we collected tissue from 34 autopsy‐derived metastases, samples rarely analyzed in previous studies. These were compared to a separate set of 17 prostatectomy specimens containing 22 foci of CaP associated with 49 examples of high‐grade prostatic intraepithelial neoplasia (PIN), a histological precursor of CaP. We compared the loss of heterozygosity (LOH) profiles of high‐grade PIN, primary CaP and metastases by analyzing 33 microsatellite markers previously found to have high frequencies of LOH in primary CaP. These markers were on chromosomes 5q, 6q, 7q, 8p, 9p, 10q, 11p, 13q, 16q, 17, 18q and 21q. In addition, markers on chromosomes 4p, 11q, 14q and 20q with no reported LOH in primary CaP were analyzed to determine the frequency of background LOH. In PIN lesions, the rate of LOH was significant only at D5S806 (20%) and D16S422 (29%). In addition, different PIN lesions within the same prostate gland were genetically diverse, indicating divergent evolution of synchronous neoplastic precursor lesions. LOH frequency was progressively higher in primary CaP and metastatic lesions. In primary CaP, significant losses occurred at the 8p, 10q, 11p, 16q, 17p, 18q and 21q loci (range 17–43%). Distinct patterns of LOH frequencies were observed in primary CaP compared with metastases. Although some loci (D16S422, D17S960, D21S156) showed similar frequencies of LOH in primary CaP and metastatic CaP, most other loci showed up to 7‐fold metastasis‐related increases. The metastatic samples revealed previously unrecognized prostate cancer LOH at D5S806, D6S262, D9S157, D13S133 and D13S227. These significant stage‐specific differences in LOH frequency specify genetic loci that may play key roles in CaP progression and could represent clinically useful biomarkers for CaP aggressiveness. Int. J. Cancer 81:219–224, 1999.

Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility.

Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to β-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of INa, If, INCX, and ICaL, respectively, and suggested their involvement in cardiac pacemaking, with ICaL being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to β-adrenergic stimulation. Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.