HLA-E-dependent presentation of Mtb-derived antigen to human CD8+ T cells.

Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.

Kinetic characterization and molecular docking of a novel, potent, and selective slow-binding inhibitor of human cathepsin L.

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC(50) of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more potent, demonstrating an IC(50) of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were k(on) = 24,000 M(-1)s(-1) and k(off) = 2.2 x 10(-5) s(-1) (K(i) = 0.89 nM). Molecular docking studies were undertaken using the experimentally derived X-ray crystal structure of papain/CLIK-148 (1cvz. pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. In addition, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC(50) of 15.4 microM and inhibited Leishmania major with an IC(50) of 12.5 microM.

Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells.

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.