No abstract

It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR⅐hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS⅐hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS⅐hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.Several transcription factors and protein kinases involved in signal transduction are recovered from cells in association with the ubiquitous heat shock protein hsp90 1 (for review, see Refs.1 and 2). These heterocomplexes with hsp90 are formed by a multicomponent chaperone machinery consisting of hsp90, hsp70, Hop, hsp40, p23, and probably also the hsp70-interacting protein Hip and the GrpE-like protein BAG-1 (for review, see Ref.3 and references therein). As first shown for the glucocorticoid receptor (GR) (4) and then for some other steroid receptors and the dioxin (Ah) receptor, association of the ligand binding domain (LBD) with hsp90 is required for the high affinity ligand binding conformation (1, 2). Complexing of the GR with hsp90 also opens up both thiol moieties (5) and trypsin cleavage sites (6, 7) in the LBD to attack by a thiol-derivatizing agent and the protease. These direct data, coupled with recent genetic observations (8), support the notion (9, 10) that the hsp90-based chaperone machinery directs an ATP-dependent partial unfolding of the receptor LBD, thus making the hydrophobic steroid-binding pocket accessible to steroid. The problem of providing access of ligands to hydrophobic binding sites situated in the interior of properly folded proteins is not unique to steroid and dioxin receptors. To test whether the hsp90-based chaperone machinery may play a more general role in opening up hydrophobic binding clefts, we have asked whether this system facilitates the...

B cell proliferation, cytokine release, and plasmablast differentiation assays CD19 + B cells were isolated from PBMCs of healthy volunteers by negative selection using the B cell purification kit II (Miltenyi Biotec,

Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of 125 I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The hemedeficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of nonfunctional enzyme.

SUMMARY The global public health community has set ambitious treatment targets to end the HIV/AIDS pandemic. With the notable absence of a cure, the goal of HIV treatment is to achieve sustained suppression of an HIV viral load, which allows for immunological recovery and reduces the risk of onward HIV transmission. Monitoring HIV viral load in people living with HIV is therefore central to maintaining effective individual antiretroviral therapy as well as monitoring progress toward achieving population targets for viral suppression. The capacity for laboratory-based HIV viral load testing has increased rapidly in low- and middle-income countries, but implementation of universal viral load monitoring is still hindered by several barriers and delays. New devices for point-of-care HIV viral load testing may be used near patients to improve HIV management by reducing the turnaround time for clinical test results. The implementation of near-patient testing using these new and emerging technologies may be an essential tool for ensuring a sustainable response that will ultimately enable an end to the HIV/AIDS pandemic. In this report, we review the current and emerging technology, the evidence for decentralized viral load monitoring by non-laboratory health care workers, and the additional considerations for expanding point-of-care HIV viral load testing.

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-kB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-kB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation. ImmunoHorizons, 2020, 4: 93-107.

Nitric-oxide synthases (NOS)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide (1-4). Nitric oxide is a signaling molecule that is involved in a variety of physiological processes, including neurotransmission, vasorelaxation, platelet aggregation, and penile erection as well as in a variety of pathological conditions including septic shock, reperfusion injury, arthritis, atherosclerosis, diabetes, and graft rejection (5-8). It has been noted (9) that because nitric oxide is not stored, released, or inactivated after synaptic release by conventional regulatory mechanisms the biosynthetic regulation of the enzyme is of great importance. For the neuronal isoform the Ca 2ϩ -mediated activation is of prime importance. However, the factors that regulate proteolytic degradation of the enzyme have not been investigated. One approach that has been successfully utilized for the study of the proteolytic turnover of other P450 cytochromes is the use of suicide inactivators (10 -13). We wished to utilize this approach for the study of NOS turnover.Suicide inactivators are chemically inert molecules that mimic the natural substrate of the enzyme and become metabolized to a highly reactive intermediate that can covalently alter important active site entities, resulting in inactivation of the enzyme (14). In effect, the compound causes the enzyme to catalyze its own demise. Because the compound must not only have affinity for the active site but also must be metabolized in a manner to generate a reactive intermediate that is in close proximity to an important active site entity, these agents have the potential to be highly specific in vivo. In addition, they react with the form of the enzyme that is engaged in catalysis and thus are useful mechanistic probes into the nature of the bioactivation reaction. In some cases, these agents covalently alter P450 cytochromes in a manner that enhances their proteolysis and turnover (10 -13, 15-17).Guanabenz, a clinically used antihypertensive agent with a guanidino moiety, was recently shown, with the use of brain and penile cytosol, to be a metabolism-based inactivator of neuronal nitric-oxide synthase (nNOS) (18). Moreover, the treatment of rats with guanabenz was found to cause not only a decrease in activity, but also a concomitant loss of immunodetectable nNOS protein. To further understand the molecular mechanisms responsible for the loss of nNOS protein in vivo, we chose to model the effects of guanabenz with the use of HEK 293 cells stably transfected with nNOS. In the current study, we have shown that guanabenz causes the enhanced proteolytic turnover of nNOS. Suicide inactivators of nNOS, such as N G -methyl-L-arginine or N 5 -(1-iminoethyl)-L-ornithine, also caused the enhanced proteolytic degradation of the enzyme, whereas the slowly reversible inhibitor N G -nitro-L-arginine or the reversible inhibitor 7-NI did not. Studies with protease inhibitors indicate that the proteasome is responsible, in part, for r...