Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes

The attachment of lysine 48 (Lys(48))-linked polyubiquitin chains to proteins is a universal signal for degradation by the proteasome. Here, we report that long Lys(48)-linked chains are resistant to many deubiquitinating enzymes (DUBs). Representative enzymes from this group, Ubp15 from yeast and its human ortholog USP7, rapidly remove mono- and diubiquitin from substrates but are slow to remove longer Lys(48)-linked chains. This resistance is lost if the structure of Lys(48)-linked chains is disrupted by mutation of ubiquitin or if chains are linked through Lys(63). In contrast to Ubp15 and USP7, Ubp12 readily cleaves the ends of long chains, regardless of chain structure. We propose that the resistance to many DUBs of long, substrate-attached Lys(48)-linked chains helps ensure that proteins are maintained free from ubiquitin until a threshold of ubiquitin ligase activity enables degradation.     

Clemastine potentiates the human P2X7 receptor by sensitizing it to lower ATP concentrations

P2X7 receptors have emerged as potential drug targets for the treatment of medical conditions such as e.g. rheumatoid arthritis and neuropathic pain. To assess the impact of pharmaceuticals on P2X7, we screened a compound library comprising approved or clinically tested drugs and identified several compounds that augment the ATP-triggered P2X7 activity in a stably transfected HEK293 cell line. Of these, clemastine markedly sensitized Ca(2+) entry through P2X7 to lower ATP concentrations. Extracellularly but not intracellularly applied clemastine rapidly and reversibly augmented P2X7-mediated whole-cell currents evoked by non-saturating ATP concentrations. Clemastine also accelerated the ATP-induced pore formation and Yo-Pro-1 uptake, increased the fractional NMDG(+) permeability, and stabilized the open channel conformation of P2X7. Thus, clemastine is an extracellularly binding allosteric modulator of P2X7 that sensitizes P2X7 to lower ATP concentrations and facilitates its pore dilation. The activity of clemastine on native P2X7 receptors, Ca(2+) entry, and whole-cell currents was confirmed in human monocyte-derived macrophages. Similar effects were observed in murine bone marrow-derived macrophages. Consistent with the data on recombinant P2X7, clemastine augmented the ATP-induced cation entry and Yo-Pro-1 uptake. In accordance with the observation that P2X7 controls the cytokine release from LPS-primed macrophages, we found that clemastine augmented the IL-1β release from LPS-primed human macrophages. Collectively, these data point to a sensitization of the recombinantly or natively expressed human P2X7 receptor toward its physiological activator, ATP, possibly leading to a modulation of macrophage-dependent immune responses.     

Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated

Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.     

Cyclin‐dependent kinase 9 forms a complex with GATA4 and is involved in the differentiation of mouse ES cells into cardiomyocytes

The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin‐dependent kinase 9 (Cdk9), a core component of positive elongation factor‐b, is a novel GATA4‐binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac‐specific Nkx2.5 promoter, were induced to differentiate on feeder‐free gelatin‐coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP‐positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant‐negative form of Cdk9 (DN‐Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6‐dichloro‐1‐β‐ribofuranosyl‐benzimidazole (DRB) on TSA‐induced myocardial cell differentiation. The introduction of the DN‐Cdk9 inhibited TSA‐induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA‐induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4. J. Cell. Physiol. 226: 248–254, 2010. © 2010 Wiley‐Liss, Inc.     

Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.     

Evidence of a novel immune responsive protein in the Hymenoptera

Honeybee populations are severely threatened by parasites and diseases. Recent outbreaks of Colony Collapse Disorder (CCD) has caused loss of more than 35% of bee colonies in the USA, and this is thought to at least in part be due to parasites and/or disease. Interestingly, the honeybee possesses of a limited set of immune genes compared to other insects. Non-canonical immune genes of honeybee are of interest because they may provide greater insights into the peculiar nature of the immune system of this social insect. Previous analyses of bee haemolymph upon bacterial challenge identified a novel leucine-rich repeat protein termed IRP30. Here we show that IRP30 behaves as a typical secreted immune protein. It is expressed simultaneously with carboxylesterase upon treatment with bacteria or other elicitors of immune response. Furthermore we characterize the gene and the mRNA encoding this protein and the IRP30 protein itself. Its regulation and evolution reveal that IRP30 belongs to a protein family, distributed broadly among Hymenoptera, suggesting its ancient function in immune response. We document an interesting case of a recent IRP30 loss in the ant Atta cephalotes and hypothesize that a putative IRP30 homolog of Nasonia emerged by convergent evolution rather than diverged from a common ancestor.     

Efficient display of two enzymes on filamentous phage using an improved signal sequence

Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50-fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.     

Rapid parallel mutation scanning of gene fragments using a microelectronic protein–DNA chip format

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.     

High Immune Response Rates and Decreased Frequencies of Regulatory T Cells in Metastatic Renal Cell Carcinoma Patients after Tumor Cell Vaccination

Our previously reported phase I clinical trial with the allogeneic gene–modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T_H1/T_H2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.     

Pathogenesis of precocious puberty in hypothalamic hamartoma

Astroglial-derived factors, as transforming growth factor (TGF)alpha and TGFbeta, act in the hypothalamus to activate luteinizing hormone-releasing hormone (LHRH) secretion. Hypothalamic hamartomas (HHs) contain normal nervous tissue in a heterotopic location. When symptomatic, they cause precocious puberty and/or characteristic gelastic seizures. Thus far, the pathogenesis of these alterations remains unknown. By examining two HHs associated with sexual precocity, we found that they contained astroglial cells expressing TGFalpha, but no LHRH neurons. In a third patient with HH, only epilepsy was present, but precocious puberty developed shortly after surgery, probably as a consequence of a surgery-induced lesion. These results imply that some HHs induce sexual precocity by activating endogenous LHRH secretion via astroglial-derived factors.