In CD28-costimulated human naïve CD4+ T cells, I-κB kinase controls the expression of cell cycle regulatory proteins via interleukin-2-independent mechanisms.

Stimulation of naïve CD4(+) T cells through engagement of the T-cell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signalling via the IL-2 receptor. However, several studies indicate that in CD28-costimulated T cells additional IL-2-independent signals are also required for cell proliferation. In this study, using a neutralizing anti-human IL-2 antibody and two selective, structurally unrelated, cell-permeable I-κB kinase (IKK) inhibitors, BMS-345541 and PS-1145, we show that in human naïve CD4(+) T cells stimulated through a short engagement of the TCR and the CD28 co-receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase-associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms.

The HIV protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse transcriptase inhibitors, adversely affect human proteasome function.

BACKGROUND: In HIV-infected patients some clinical and immunological benefits of antiretroviral therapy, which frequently include a combination of HIV protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs), cannot be solely explained by the drugs' action on viral enzymes. Proteasomes constitute the central protease of the ubiquitin ATP-dependent pathway involved in many cellular processes, as well as in HIV maturation and aggressiveness. OBJECTIVE: To explore whether the PIs nelfinavir and saquinavir and the RTIs abacavir, nevirapine, delavirdine, stavudine and didanosine affect proteasome function in vitro and in vivo. METHODS: Peptidase activity of purified human 26S and 20S proteasomes was assayed with and without the drugs at different concentrations. Intracellular proteasome proteolytic activity was evaluated by searching for ubiquitin-tagged proteins in HL60 cells incubated with and without the drugs. RESULTS: At therapeutic dosages, nelfinavir and saquinavir inhibited proteasome peptidase activity and caused intracellular accumulation of polyubiquitinated proteins, a hallmark of proteasome proteolytic inhibition in vivo; the RTIs failed to evoke either effect. CONCLUSION: Proteasomes are targeted by the two PIs but not the RTIs. Therefore, in HIV-infected patients the beneficial effect of a therapy including one of the two PIs should partly rely on inhibition of host proteasome function.

Down-regulation of pyruvate dehydrogenase phosphatase in obese subjects is a defect that signals insulin resistance.

OBJECTIVE: The objective of this study was to determine whether down-regulation of pyruvate dehydrogenase phosphatase (PDP) is responsible for poorly active pyruvate dehydrogenase (PDH) in circulating lymphocytes (CLs) of obese subjects (ObS), and if so, whether it improves when their plasma insulin rises. RESEARCH METHODS AND PROCEDURES: PDH activity was compared in lysed CLs of 10 euglycemic ObS and 10 sex- and age-matched controls before and during plasma insulin enhancement in an oral glucose tolerance test. It was evaluated without (PDHa) or with Mg/Ca or Mg at various concentrations to assess PDP1 or PDP2 activities or with Mg/Ca and exogenous PDP to determine total PDH activity (PDHt), which is an indirect measure of the amount of PDH. The insulin sensitivity index was calculated, and PDP1 and PDP2 mRNA was sought in the CLs. RESULTS: At T0 in ObS, PDHt was normal, whereas PDHa and PDP1 activity was below normal at all Mg/Ca concentrations. PDP2 activity was undetectable in both groups. PDP1 and PDP2 mRNA was identified, and insulin sensitivity index and PDHa were directly correlated. During the oral glucose tolerance test, plasma insulin rose considerably more in ObS than in controls; PDHa and PDP1 activity also increased but remained significantly below normal, and PDHt was unvaried in both groups. DISCUSSION: PDP1 is down-regulated in CLs of ObS because it is poorly sensitive to Mg/Ca; this defect is attenuated when plasma insulin is greatly enhanced.

The human 26S proteasome is a target of antiretroviral agents.

BACKGROUND: Proteasomes constitute the degradative machinery of the ubiquitin/adenosine triphosphate-dependent proteolytic pathway, which is involved in many cell functions, including immune response and apoptosis, and in HIV maturation and infectivity. OBJECTIVE: To examine whether proteasomes are targeted by antiretroviral agents. METHODS: Chymotrypsin-like, trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of purified human 26S and 20S proteasomes, the latter depleted or enriched in 11S regulator, were assayed after incubation with indinavir, lamivudine and zidovudine at 1-80 microM alone and in combination. To assess the drug effects on cellular functions regulated by proteasomes, the accumulation of ubiquitin-tagged proteins, the processing of the nuclear factor kappa B precursor p105, and the degradation of the inhibitor of nuclear factor kappa B, isoform alpha (IkappaBalpha) were evaluated by Western immunoblotting in Jurkat cells after incubation for 6 h with the drugs above. RESULTS: Trypsin-like and mostly chymotrypsin-like activities of purified 26S proteasome were inhibited by each drug from 10 to 80 microM, more by double combinations and mostly by the triple combination. The peptidyl-glutamyl-peptide hydrolysing activity of the 26S proteasome and the three peptidase activities of the 20S proteasome, depleted or enriched in 11S regulator, were unaffected. The accumulation of ubiquitin-tagged proteins, reduced IkappaBalpha degradation and p105 processing were appreciable in intact cells with the triple drug combination. CONCLUSION: The human 26S proteasome is a target of antiretroviral agents. This suggests that the antiviral action and some clinical and immunological benefits of combined antiretroviral therapy rely not only on its known effects on viral enzymes, but also on host cell components.