Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK

Altered Thymic Function during Interferon Therapy in HCV-Infected Patients

Interferon alpha (IFNα) therapy, despite good efficacy in curing HCV infection, leads to major side effects, in particular inducement of a strong peripheral T-cell lymphocytopenia. We here analyze the early consequences of IFNα therapy on both thymic function and peripheral T-cell homeostasis in patients in the acute or chronic phase of HCV-infection as well as in HIV/HCV co-infected patients. The evolution of T-cell subsets and T-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of T-cell receptor excision circles (TREC) and estimation of intrathymic precursor T-cell proliferation during the first four months following the initiation of IFNα therapy. Beginning with the first month of therapy, a profound lymphocytopenia was observed for all T-cell subsets, including naïve T-cells and recent thymic emigrants (RTE), associated with inhibition of intrathymic precursor T-cell proliferation. Interleukin (IL)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. This was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble CD127. Decrease in IL-7 plasma concentration under IFNα therapy correlated with the decline in HCV viral load, thymic activity and RTE concentration in blood. These data demonstrate that IFNα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs T-cell homeostasis. Such a side effect might be detrimental for the continuation of IFNα therapy and may lead to an increased level of infectious risk, in particular in HIV/HCV co-infected patients. Altogether, this study suggests the therapeutic potential of IL-7 in the maintenance of peripheral T-cell homeostasis in IFNα-treated patients

Aged PROP1 Deficient Dwarf Mice Maintain ACTH Production

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1null (Prop1-/-) and the Ames dwarf (Prop1df/df) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism

Zinc Coordination Is Required for and Regulates Transcription Activation by Epstein-Barr Nuclear Antigen 1

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential

A genome-wide screen of Epstein-Barr virus proteins that modulate host SUMOylation identifies a SUMO E3 ligase conserved in herpesviruses

<div><p>Many cellular processes pertinent for viral infection are regulated by the addition of small ubiquitin-like modifiers (SUMO) to key regulatory proteins, making SUMOylation an important mechanism by which viruses can commandeer cellular pathways. Epstein-Barr virus (EBV) is a master at manipulating of cellular processes, which enables life-long infection but can also lead to the induction of a variety of EBV-associated cancers. To identify new mechanisms by which EBV proteins alter cells, we screened a library of 51 EBV proteins for global effects on cellular SUMO1 and SUMO2 modifications (SUMOylation), identifying several proteins not previously known to manipulate this pathway. One EBV protein (BRLF1) globally induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of promeylocytic leukemia nuclear bodies. However, unlike its homologue (Rta) in Kaposi’s sarcoma associated herpesvirus, it did not appear to have ubiquitin ligase activity. In addition we identified the EBV SM protein as globally upregulating SUMOylation and showed that this activity was conserved in its homologues in herpes simplex virus 1 (HSV1 UL54/ICP27) and cytomegalovirus (CMV UL69). All three viral homologues were shown to bind SUMO and Ubc9 and to have E3 SUMO ligase activity in a purified system. These are the first SUMO E3 ligases discovered for EBV, HSV1 and CMV. Interestingly the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins, and the conservation of this SUMO E3 ligase activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.</p></div

Effect of SM, UL54 and UL69 on global SUMO1 and SUMO2 modifications.

<p>293T (A) and CNE2Z (C) cells were co-transfected with plasmids expressing His6-SUMO1 (left panels) or His6-SUMO2 (right panels) and either ICP0, LMP1, FLAG-SM, FLAG-UL54, FLAG-UL69 or empty vector control. HeLa cells (B) stably expressing His6-SUMO1 (left panels) or His6-SUMO2 (right panels) were transfected with plasmids expressing ICP0, LMP1, FLAG-SM, FLAG-UL54, FLAG-UL69 or empty vector control. His6-tagged proteins were recovered as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007176#ppat.1007176.g001" target="_blank">Fig 1</a> and immunoblotted for SUMO1 or SUMO2 as indicated. Samples of the input lysates were also immunoblotted for actin and FLAG.</p

Screen of EBV Lytic proteins for global effects on cellular SUMOylation^*.

<p>Screen of EBV Lytic proteins for global effects on cellular SUMOylation^{<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007176#t001fn001" target="_blank">*</a>}.</p