Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

Limestone gravel as growth medium in hydroponics

Summary Plants were grown in gravel beds of basalt or limestone. With the standard nutrient solution yields for the plants grown in the limestone gravel were very much reduced and the plants were chlorotic. This chlorosis and reduced yields remained even if the gravel was acid or water washed beforehand, or if it was pretreated in high phosphate concentrations.High yields, comparable to those obtained in basalt beds were obtained if phosphate and iron were added in small amounts (0.1mM P, 1 mg/1 Fe) at each irrigation. This technique to avoid lime induced chlorosis can be easily and economically accomplished in hydroponics. It proved successful here with lettuce, cucumber, tomato and eggplants.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

Cell-type action specificity of auxin on Arabidopsis root growth

The plant hormone auxin plays a critical role in root growth and development; however, the contributions or specific roles of cell-type auxin signals in root growth and development are not well understood. Here, we mapped tissue and cell types that are important for auxin-mediated root growth and development by manipulating the local response and synthesis of auxin. Repressing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele strongly inhibited root growth, with the largest effect observed in the endodermis. Enhancing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele also caused reduced root growth, albeit to a lesser extent. Moreover, we established that root growth was inhibited by enhancement of auxin synthesis in specific cell types of the epidermis, cortex and endodermis, whereas increased auxin synthesis in the pericycle and stele had only minor effects on root growth. Our study thus establishes an association between cellular identity and cell type-specific auxin signaling that guides root growth and development.     

The Effect of Proteasome Inhibition on the Generation of the Human Leukocyte Antigen (HLA) Peptidome

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.The repertoires and levels of peptides, presented by the major histocompatibility complex (MHC)¹ class I molecules at the cells'' surface, are modulated by multiple factors. These include the rates of synthesis and degradation of their source proteins, the transport efficacy of the peptides through the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), their subsequent processing and loading onto the MHC molecules within the ER, and the rates of transport of the MHC molecules with their peptide cargo to the cell surface. The off-rates of the presented peptides, the residence time of the MHC complexes at the cell surface, and their retrograde transport back into the cytoplasm, influence, as well, the presented peptidomes (reviewed in (1)). Even though significant portions of the MHC class I peptidomes are thought to be derived from newly synthesized proteins, including misfolded proteins, defective ribosome products (DRiPs), and short lived proteins (SLiPs), most of the MHC peptidome is assumed to originate from long-lived proteins, which completed their functional cellular roles or became defective (retirees), (reviewed in (2)).The main protease, supplying the MHC peptidome production pipeline, is thought to be the proteasome (3). It is also responsible for generation of the final carboxyl termini of the MHC peptides (4), (reviewed in (5)). The final trimming of the n-termini of the peptides, within the endoplasmic reticulum (ER), is thought to be performed by amino peptidases, such as ERAP1/ERAAP, which fit the peptides into their binding groove on the MHC molecules (6) (reviewed in (7)). Nonproteasomal proteolytic pathways were also suggested as possible contributors to the MHC peptidome, including proteolysis by the ER resident Signal peptide peptidase (8, 9), the cytoplasmic proteases Insulin degrading enzyme (10), Tripeptidyl peptidase (11–13), and a number of proteases within the endolysosome pathway (reviewed recently in (14–17)). In contrast to the mostly cytoplasmic and ER production of the MHC class I peptidome, the class II peptidome is produced in a special compartment, associated with the endolysosome pathway (18–20). This pathway is also thought to participate in the cross presentation of class I peptides, derived from proteins up-taken by professional antigen presenting cells (21), (reviewed in (15–17, 22)).The centrality of the proteasomes in the generation of the MHC peptidome was deduced mostly from the observed change in presentation levels of small numbers of selected peptides, following proteasome inhibition (3, 23). Even the location of some of the genes encoding the catalytic subunits of the immunoproteasome (LMP2 and LMP7) (24) within the MHC class II genomic locus, was suggested to support the involvement of the proteasome in the generation of the MHC class I peptidome (25). Similar conclusions were deduced from alterations in peptide presentation, following expression of the catalytic subunits of the immunoproteasome (26), (reviewed in (5)). Yet, although most of the reports indicated reductions in presentation of selected peptides by proteasome inhibition (3, 27–29), others have observed only limited, and sometimes even opposite effects (23, 30–32).The matter is further complicated by the indirect effects of proteasome inhibition used for such studies on the arrest of protein synthesis by the cells (33–35), on the transport rates of the MHC molecules to the cell surface, and on their retrograde transport back to the vesicular system (36) (reviewed in (37)). Proteasome inhibition likely causes shortage of free ubiquitin, reduced supply of free amino acids, and induces an ER unfolded protein response (UPR), which signals the cells to block most (but not all) cellular protein synthesis (reviewed in (38)). Because a significant portion of the MHC peptidome originates from degradation of DRiPs and SLiPs (reviewed in (2)), arrest of new protein synthesis should influence the presentation of their derived MHC peptides. Taken together, these arguments may suggest that merely following the changes in the presentation levels of the MHC molecules, or even of specific MHC peptides, after proteasome inhibition, does not provide the full picture for deducing the relative contribution of the proteasomal pathway to the production of the MHC peptidome (reviewed in (7)).MHC peptidome analysis can be performed relatively easily by modern capillary chromatography combined with mass spectrometry (reviewed in (39)). The peptides are recovered from immunoaffinity purified MHC molecules after detergent solubilization of the cells (40, 41), from soluble MHC molecules secreted to the cells'' growth medium (42, 43) or from patients'' plasma (44). The purified peptides pools are resolved by capillary chromatography and the individual peptides are identified and quantified by tandem mass spectrometry (40), (reviewed in (45–47)). In cultured cells, quantitative analysis can also be followed by metabolic incorporation of stable isotope labeled amino acids (SILAC) (48). Furthermore, the rates of de novo synthesis of both MHC peptides and their proteins of origin can be followed using the dynamic-SILAC proteomics approach (49) with its further adaptation to HLA peptidomics (50–52).This study attempts to define the relative contribution of the proteasomes to the production of HLA class I peptidome by simultaneously following the effects of proteasome inhibitors, epoxomicin and bortezomib (Velcade), on the rates of de novo synthesis of both the HLA class I peptidome and the cellular proteome of the same MCF-7 human breast cancer cultured cells. The proteasome inhibitors did not reduce the levels of HLA presentations, yet affected the rates of production of both the HLA peptidome and cellular proteome, mostly decreasing, but also increasing some of the synthesis rates of the HLA peptides and cellular proteins. Thus, we suggest that the degree of contribution of the proteasomal pathway to the production of the HLA-I peptidome should be re-evaluated in accordance with their effects on the entire HLA class-I peptidome, while taking into consideration the inhibitors'' effects on the synthesis (and degradation) rates of the source proteins of each of the studied HLA peptides.     

Neuronal activity preceding directional and nondirectional cues in the premotor cortex of rhesus monkeys

Pre-cue activity, the neuronal modulation that precedes a predictable stimulus, was studied in the premotor cortex of three rhesus monkeys. In one condition, a directional cue dictated the timing and target of a forelimb movement. In another condition, a nondirectional cue provided identical timing information but did not indicate the target. Of 501 task-related neurons recorded in premotor cortex, 168 showed pre-cue activity. The onset time of pre-cue activity varied markedly from trial to trial and cell to cell, ranging from trial initiation to 4.8 sec later. No pre-cue activity reflected the direction of limb movement; thus, the data argue against the hypothesis that pre-cue activity reflects preparation for specific limb movements. A small number of cells showed greater pre-cue activity before directional than before nondirectional cues, and this difference may reflect anticipation of the cue's directional information. However, the vast majority (84%) of neurons lacked such differences. We therefore hypothesize that most pre-cue activity reflects or contributes to a facet of behavior common to the two conditions: anticipation of the time and/or nature of events.