Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B−/− or Cat D−/− antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B−/− splenocytes, as it did in Cat D−/− cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca2+ influx, increase Ca2+/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA2 were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA2 and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA2 by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA2 activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIα subunit would reveal those tissues and signaling events that require anchored PKA. RIIα knockout mice appear normal and healthy. In adult skeletal muscle, RIα protein levels increased to partially compensate for the loss of RIIα. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIα knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA–AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIα subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIα subunit. The potentiation of the L-type Ca2+ channel in RIIα knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIα is capable of physiologically relevant anchoring interactions.

Aminopeptidase A inhibitors as potential central antihypertensive agents

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT1) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT1 receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca2+ concentration and is expressed as a long-lasting increase of postsynaptic GABAA receptor sensitivity. Herein we show that inhibitors of the Ca2+/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca2+ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Replicative helicases can translocate through abasic site-induced covalent topoisomerase IV–DNA complexes

Some topoisomerase inhibitors trap covalent topoisomerase–DNA complexes as topoisomerase–drug–DNA ternary complexes. Ternary complex formation results in inhibition of DNA replication and generation of permanent double-strand breaks. Recent demonstrations of the stimulation of covalent topoisomerase–DNA complex formation by DNA lesions suggest that DNA damage may act as an endogenous topoisomerase poison. We have investigated the effects of abasic (AP) sites on topoisomerase IV (Topo IV). AP sites can stimulate the formation of covalent Topo IV–DNA complexes when they are located either within the 4 base overhang generated by DNA scission or immediately 5′ to the point of scission (the –1 position). Thus, the AP site acts as a position-specific, endogenous topoisomerase poison. Both EDTA and salt can reverse covalent Topo IV–DNA complexes induced by AP sites located within the 4 base overhang. Interestingly, an AP site at the –1 position inhibits EDTA-mediated reversal of formation of the covalent Topo IV–DNA complex. Furthermore, we find that, unlike quinolone-induced covalent Topo IV–DNA complexes, AP site-induced covalent Topo IV–DNA complexes do not inhibit the helicase activities of the DnaB and T7 Gene 4 proteins. These results suggest that the AP site-induced poisoning of Topo IV does not arrest replication fork progression.

Mechanism of topology simplification by type II DNA topoisomerases

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

A type IB topoisomerase with DNA repair activities

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase β. Second, on the basis of sequence homology between topo V and polymerase β, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5′ 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3′-hydroxyl and 5′-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.

The structure of the human βII-tryptase tetramer: Fo(u)r better or worse

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-Å crystal structure of human βII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 Å × 15 Å govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii1

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Single-strand interruptions in replicating chromosomes cause double-strand breaks

Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends. To model replication fork collapse in vivo, I constructed phage λ chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates Escherichia coli cells, producing M13 nicking enzyme. I detected double-strand breaks at the nicking sites in λ DNA purified from these cells. The double-strand breakage depends on (i) the presence of the nicking site; (ii) the production of the nicking enzyme; and (iii) replication of the nick-containing chromosome. Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.

Excitotoxicity in the lung: N-methyl-D-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase.

Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the "adult respiratory distress syndrome," and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

Topoisomerase IV is a target of quinolones in Escherichia coli.

We have demonstrated that, in Escherichia coli, quinolone antimicrobial agents target topoisomerase IV (topo IV). The inhibition of topo IV becomes apparent only when gyrase is mutated to quinolone resistance. In such mutants, these antibiotics caused accumulation of replication catenanes, which is diagnostic of a loss of topo IV activity. Mutant forms of topo IV provided an additional 10-fold resistance to quinolones and prevented drug-induced catenane accumulation. Drug inhibition of topo IV differs from that of gyrase. (i) Wild-type topo IV is not dominant over the resistant allele. (ii) Inhibition of topo IV leads to only a slow stop in replication. (iii) Inhibition of topo IV is primarily bacteriostatic. These differences may result from topo IV acting behind the replication fork, allowing for repair of drug-induced lesions. We suggest that this and a slightly higher intrinsic resistance of topo IV make it secondary to gyrase as a quinolone target. Our results imply that the quinolone binding pockets of gyrase and topo IV are similar and that substantial levels of drug resistance require mutations in both enzymes.