A cross-sectional study on biochemical parameters of bone turnover and vitamin d metabolites in healthy dutch children and young adults

AIM: To provide reference data of biochemical markers of bone turnover and vitamin D metabolites for children and young adults. METHODS: Blood samples were taken from 176 healthy Dutch children and young adults (age range 7.6-25.3 years) to assess serum calcium, alkaline phosphatase, inorganic phosphate, osteocalcin, collagen type I cross-linked N-telopeptide, N-terminal propeptide of type I procollagen, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 levels. Cross-linked telopeptide of type I collagen and carboxy-terminal propeptide of type I procollagen were assessed in 286 subjects (age range 1.4-25.3 years). RESULTS: Calcium and vitamin D levels were independent of age. The peak concentrations for collagen type I cross-linked N-telopeptide, cross-linked telopeptide of type I collagen, carboxy-terminal propeptide of type I procollagen, N-terminal propeptide of type I procollagen, alkaline phosphatase, and osteocalcin were found during puberty, in girls approximately 2.5 years earlier than in boys. Strong correlations were found between the markers of bone turnover, while no correlation was found between the markers of bone turnover and bone mineral density measured by dual-energy X-ray absorptiometry. CONCLUSIONS: Single measurements of bone markers cannot predict bone density. Reference data according to gender, age, and Tanner stage are given which allow calculating standard deviation scores adjusted for age and gender.     

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.     

Alternatives to donor matching for control of graft-versus-host disease

Graft-versus-host disease (GvHD) after bone-marrow transplantation in dogs is controlled by many different genetic systems. In littermate combinations identical for the major histocompatibility complex (MHC) the number of systems that influence GvHD is related to the number of donor lymphocytes injected. If the number of donor lymphocytes administered is sufficiently low, minor histocompatibility systems do not influence survival after bone-marrow transplantation. With increasing numbers of donor lymphocytes the beneficial influence of MHC matching on GvH incidence and severity disappears and minor histocompatibility antigens, coded for on at least two other autosomal chromosomes as well as possibly the Y chromosome, can cause severe GvHD. In contrast, the X chromosome does not appear to carry a histocompatibility system that is of relevance to GvHD control. The severity and tissue distribution of histological signs of GvHD in recipients of bone-marrow and lymph-node cells from MHC-identical donors are similar to those in recipients of MHC-mismatched bone-marrow cells. Female donors do appear to cause severe GvHD more frequently than males. In contrast to rhesus monkey and human bone-marrow cells, dog bone-marrow cells are negative in PHA tests. This is in accordance with the generally benign course of GvHD in dogs that are treated with bone-marrow cells only from histocompatible littermate donors. The influence of the sex of the bone-marrow donor on GvHD incidence and severity is not reflected in differences between PHA tests with male and female dog lymphocytes. A better predictive test for GvH potential than the PHA test appears to be needed. Alternatives to additional donor selection for the prevention of GvHD in histocompatible recipients appear to be the use of a male donor and the removal of lymphocytes from bone-marrow-cell suspensions prior to transplantation.     

PTPBR7 binding proteins in myelinating neurons of the mouse brain

Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may influence PTPBR7 activity, however, remain to be determined. We here show that the PTPBR7 extracellular domain binds to highly myelinated regions in mouse brain, in particular the white matter tracks in cerebellum. PTPBR7 deficiency does not alter this binding pattern, as witnessed by RAP in situ staining of Ptprr^-/- mouse brain sections. Additional in situ and in vitro experiments also suggest that sugar moieties of heparan sulphate and chondroitin sulphate glycosaminoglycans are not critical for PTPBR7 binding. Candidate binding proteins were affinity-purified exploiting the PTPBR7 extracellular domain and identified by mass spectrometric means. Results support the suggested link between PTPRR isoforms and cerebellar calcium ion homeostasis, and suggest an additional role in the process of cell-cell adhesion.     

The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel

The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.     

Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies

Neurodegenerative disorders such as Huntington’s disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin–proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington’s disease, and support the reported absence of proteasome impairment in mouse models of Huntington’s disease.     

T cell recognition of naturally presented epitopes of self-heat shock protein 70

Self-reactive T cells have shown to have a potential role as regulators of the immune system preventing or even suppressing autoimmunity. One of the most abundant proteins that can be eluted from human HLA molecules is heat shock protein 70 (HSP70). The aims of the current study are to identify HSP70 epitopes based on published HLA elution studies and to investigate whether T cells from healthy individuals may respond to such self-epitopes. A literature search and subsequent in silico binding prediction based on theoretical MHC binding motifs resulted in the identification of seven HSP70 epitopes. PBMCs of healthy controls proliferated after incubation with two of the seven peptides (H167 and H290). Furthermore H161, H290, and H443 induced CD69 expression or production of cytokines IFNγ or TNFα in healthy controls. The identification of these naturally presented epitopes and the response they elicit in the normal immune system make them potential candidates to study during inflammatory conditions as well as in autoimmune diseases.     

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.