Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena

Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression. The roles of other covalent histone modifications remain poorly understood. To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena, yeast, and human HeLa cells. In Tetrahymena, transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3. Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast. These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone. In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena, we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined. Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Antigen-driven CD4+ T cell and HIV-1 dynamics: Residual viral replication under highly active antiretroviral therapy

Antigen-induced stimulation of the immune system can generate heterogeneity in CD4+ T cell division rates capable of explaining the temporal patterns seen in the decay of HIV-1 plasma RNA levels during highly active antiretroviral therapy. Posttreatment increases in peripheral CD4+ T cell counts are consistent with a mathematical model in which host cell redistribution between lymph nodes and peripheral blood is a function of viral burden. Model fits to patient data suggest that, although therapy reduces HIV replication below replacement levels, substantial residual replication continues. This residual replication has important consequences for long-term therapy and the evolution of drug resistance and represents a challenge for future treatment strategies.

Chemical complementation identifies a proton acceptor for redox-active tyrosine D in photosystem II

Through the use of site-directed mutagenesis and chemical rescue, we have identified the proton acceptor for redox-active tyrosine D in photosystem II (PSII). Effects of chemical rescue on the tyrosyl radical were monitored by EPR spectroscopy. We also have acquired the Fourier–transform infrared (FT-IR) spectrum associated with the oxidation of tyrosine D and concomitant protonation of the acceptor. Mutant and isotopically labeled PSII samples are used to assign vibrational lines in the 3,600–3,100 cm−1 region to N-H modes of His-189 in the D2 polypeptide. When His-189 in D2 is changed to a leucine (HL189D2) in PSII, dramatic alterations of both EPR and FT-IR spectra are observed. When imidazole is introduced into HL189D2 samples, results from both EPR and FT-IR spectroscopy argue that imidazole is functionally reconstituted into an accessible pocket and that imidazole acts as a chemical mimic for His-189. Small perturbations of EPR and FT-IR spectra are consistent with access to this pocket in wild-type PSII, as well. Structures of the analogous site in bacterial reaction centers suggest that an accessible pocket, large enough to contain imidazole, is bordered by tyrosine D and His-189 in the D2 polypeptide. These data provide evidence that His-189 in the D2 polypeptide of PSII acts as a proton acceptor for redox-active tyrosine D and that proton transfer to the imidazole ring facilitates the efficient oxidation/reduction of tyrosine D.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the α-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

Active galactic nuclei

Active galactic nuclei are the most powerful, long-lived objects in the Universe. Recent data confirm the theoretical idea that the power source is accretion into a massive black hole. The common occurrence of obscuration and outflows probably means that the contribution of active galactic nuclei to the power density of the Universe has been generally underestimated.

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Functional dynamics in the active site of the ribonuclease binase

Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-μs time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a β-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access/egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum kcat (1,500 s−1) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover.

Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process.

Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

Optical evidence for the unification of active galactic nuclei and quasi-stellar objects.

There is a variety of optical evidence for some unification of different types of active galactic nuclei and quasi-stellar objects (QSOs). The case is very strong for the unification of at least some Seyfert galaxies, where polarization data show that the type assigned to the Seyfert galaxy must depend on viewing direction. It has been proposed that Fanaroff-Riley type 2 (FR2) radio galaxies are quasars seen in a direction from which the quasar is obscured, and there is some limited direct evidence for this picture. The broad absorption line QSOs may be normal QSOs seen from a special direction. Some of the sources observed to have high luminosities in the far infrared could be obscured QSOs and active nuclei. Mergers and interactions are likely to play an important role in nuclear activity, and active galaxies and QSOs could change their apparent types through these encounters followed by subsequent evolution.

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

Volunteer Engagement: Does Engagement Predict the Degree of Satisfaction among New Volunteers and the Commitment of Those who have been Active Longer?

This study examines the concept of engagement in samples of volunteers from different non-profit organisations. Study 1 analyzes the psychometric properties of the abbreviated version of the Utrecht Work Engagement Scale (UWES) (Schaufeli, Bakker, & Salanova, 2006a). Two factorial structures are examined: one-dimensional and three-dimensional structures. Based on the Three-Stage Model of Volunteers’ Duration of Service (Chacón, Vecina, & Dávila, 2007), Study 2 investigates the relationship between engagement, volunteer satisfaction, and intention to remain in a sample of new volunteers and the relationship between engagement, organisational commitment, and intention to remain in a sample of veteran volunteers. Moderated mediation analysis is provided using duration of service as a moderator in order to set a splitting point between new and veteran volunteers. The results of the confirmatory factor analysis suggest that the three-factor model fits better to the data. Regarding the structural models, the first one shows that engagement is crucial to volunteer satisfaction during the first stage, while volunteer satisfaction is the key variable in explaining intention to continue. The second structural model shows that engagement reinforces the participant’s commitment to the organisation, while organizational commitment predicts intention to continue. Both models demonstrate a notable decline when samples are changed.

The magnetically-active, low-mass, triple system WDS 19312+3607 (Research Note)

Aims. We investigated in detail the system WDS 19312+3607, whose primary is an active M4.5Ve star previously inferred to be young (τ ~ 300–500 Ma) based on its high X-ray luminosity. Methods. We collected intermediate- and low-resolution optical spectra taken with 2 m-class telescopes, photometric data from the B to 8 μm bands, and data for eleven astrometric epochs with a time baseline of over 56 years for the two components in the system, G 125–15 and G 125–14. Results. We derived the M4.5V spectral types of both stars, confirmed their common proper motion, estimated their heliocentric distance and projected physical separation, determined their Galactocentric space velocities, and deduced a most-probable age of older than 600 Ma. We discovered that the primary, G 125–15, is an inflated, double-lined, spectroscopic binary with a short period of photometric variability of 1.6 d, which we associated with orbital synchronisation. The observed X-ray and Hα emissions, photometric variability, and abnormal radius and effective temperature of G 125–15 AB are indicative of strong magnetic activity, possibly because of the rapid rotation. In addition, the estimated projected physical separation between G 125–15 AB and G 125–14 of about 1200 AU ensures that WDS 19312+3607 is one of the widest systems with intermediate M-type primaries. Conclusions. G 125–15 AB is a nearby (d ≈ 26 pc), bright (J ≈ 9.6 mag), active spectroscopic binary with a single proper-motion companion of the same spectral type at a wide separation. They are thus ideal targets for specific follow-ups to investigate wide and close multiplicity or stellar expansion and surface cooling because of the lower convective efficiency.

DUI program as OEF/OIF Veterans Outreach: Descriptive Data from Two Community-Based Outpatient Clinics

Although initially conceived as providing simply the preventive portion of an extended continuum of care for veterans, the Driving Under the Influence (DUI) program has turned out to be an important outreach service for active duty or recently discharged OEF/OIF (Operation Enduring Freedom/Operation Iraqi Freedom) veterans. Veterans receive empirically-based, state-mandated education and therapy under the only Department of Veterans Affairs (VA) - sponsored DUI program in the State of Colorado, with the advantage of having providers who are sensitive to symptoms of Post-Traumatic Stress Disorder (PTSD) and other relevant diagnoses specific to this population, including Traumatic Brain Injury (TBI). In this paper, the rapid growth of this program is described, as well as summary data regarding the completion, discontinuation, and augmentation of services from the original referral concern. Key results indicated that for nearly one third (31.9%) of the OEF/OIF veterans who were enrolled in the DUI program, this was their initial contact with the VA health care system. Furthermore, following their enrollment in the DUI program, more than one fourth (27.6%) were later referred to and attended other VA programs including PTSD rehabilitation and group therapy, anger management, and intensive inpatient or outpatient dual diagnosis programs. These and other findings from this study suggest that the DUI program may be an effective additional pathway for providing treatment that is particularly salient to the distinctive OEF/OIF population; one that may also result in earlier intervention for problem drinking and other problems related to combat. Relevant conclusions discussed herein primarily aim to improve providers' understanding of effective outreach, and to enhance the appropriate linkages between OEF/OIF veterans and existing VA services.