NF-KB protein purification from bovine spleen: Nucleotide stimulation and binding site specificity

The activity of the enhancer for the κ immunoglobulin light chain gene critically depends on the presence in the nucleus of the NF-κB protein. We purified NF-κB over 50,000-fold and identified two protein species, 42 and 44 kDa, that could be eluted and renatured from a sodium dodecyl sulfate/polyacrylamide gel to give specific DNA-binding activity. Binding of the purified bovine NF-κB as well as that from human and murine B- or T-lymphoid cell extracts was dramatically stimulated by nucleoside triphosphates. This effect distinguished NF-κB from a related factor, H2-TF1. Purified NF-κB interacted efficiently with regulatory sequences that function during either B- or T-lymphocyte activation, including the human immunodeficiency virus enhancer and a NF-κB binding site we detected in the interleukin 2 enhancer

The control of CD4+CD25+Foxp3+ regulatory T cell survival

CD4+CD25+Foxp3+ regulatory T (Treg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that Treg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of Treg cells in Bim-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25+ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in Treg cells. Our study reveals that the survival of Treg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate Treg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate Treg cells that could be utilized for their therapeutic applications

30 Years of NF-κB: A Blossoming of Relevance to Human Pathobiology

NF-κB was discovered 30 years ago as a rapidly inducible transcription factor. Since that time, it has been found to have a broad role in gene induction in diverse cellular responses, particularly throughout the immune system. Here, we summarize elaborate regulatory pathways involving this transcription factor and use recent discoveries in human genetic diseases to place specific proteins within their relevant medical and biological contexts

30 Years of NF-κB: A Blossoming of Relevance to Human Pathobiology

NF-κB was discovered 30 years ago as a rapidly inducible transcription factor. Since that time, it has been found to have a broad role in gene induction in diverse cellular responses, particularly throughout the immune system. Here, we summarize elaborate regulatory pathways involving this transcription factor and use recent discoveries in human genetic diseases to place specific proteins within their relevant medical and biological contexts

The Molecular Mechanisms of Regulatory T Cell Immunosuppression

CD4+CD25+Foxp3+ T lymphocytes, known as regulatory T cells or Tregs, have been proposed to be a lineage of professional immune suppressive cells that exclusively counteract the effects of the immunoprotective “helper” and “cytotoxic” lineages of T lymphocytes. Here we discuss new concepts on the mechanisms and functions of Tregs. There are several key points we emphasize: 1. Tregs exert suppressive effects both directly on effector T cells and indirectly through antigen-presenting cells; 2. Regulation can occur through a novel mechanism of cytokine consumption to regulate as opposed to the usual mechanism of cytokine/chemokine production; 3. In cases where CD4+ effector T cells are directly inhibited by Tregs, it is chiefly through a mechanism of lymphokine withdrawal apoptosis leading to polyclonal deletion; and 4. Contrary to the current view, we discuss new evidence that Tregs, similar to other T-cells lineages, can promote protective immune responses in certain infectious contexts (Chen et al., 2011; Pandiyan et al., 2011). Although these points are at variance to varying degrees with the standard model of Treg behavior, we will recount developing findings that support these new concepts

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

HIV-1 Directly Kills CD4+ T Cells by a Fas-independent Mechanism

The mechanism by which HIV-1 induces CD4+ T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4+ T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4+ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism

14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

<p>Abstract</p> <p>Background</p> <p>Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G₂,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade.</p> <p>Results</p> <p>We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vpr_v-induced G₂,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G₂,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β.</p> <p>Conclusion</p> <p>Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins.</p> <p>Reviewers</p> <p>This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.</p

Human Immunodeficiency Virus Type 1 Vif causes dysfunction of Cdk1 and CyclinB1: implications for cell cycle arrest

The two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), inhibit cell-cycle progression at the G2 phase of the cell cycle. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was disregulated. Finally, Vif-induced cell cycle arrest was correlated with proviral expression of Vif. Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation