Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections.

The proteasome is a nuclear-cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both ß1 and ß5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 [PtoDC3000(ΔhQ)] whilst the activity profile of the ß1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced ß1 and ß5 activities to strongly suppressed ß5 activities, revealing that ß1 and ß5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.

Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses.

RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.

Discovery of a potent and highly ß1 specific proteasome inhibitor from a focused library of urea-containing peptide vinyl sulfones and peptide epoxyketones.

Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the proteasome. Compounds 13 and 15 are ß5 selective and their potency exceeds that of syringolin A. In contrast, 5 may well be the most potent ß1 selective compound active in living cells reported to date.

Two-step bioorthogonal activity-based proteasome profiling using copper-free click reagents: a comparative study.

The development and application of bioorthogonal two-step labeling techniques receives much attention. Employing bifunctional proteasome probe 2 the efficiency of two-step labeling of recently published biotinylated cyclooctynes 3-5 is compared to Staudinger-Bertozzi ligation in cell extracts and living cells. While cyclooctynes 3-5 react faster and at a much lower concentration then the Staudinger-Bertozzi benchmark, background labeling is considerable with these reagents.

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.

Synthesis of L-altro-1-deoxynojirimycin, D-allo-1-deoxynojirimycin, and D-galacto-1-deoxynojirimycin from a single chiral cyanohydrin.

The chemoenzymatic synthesis of three 1-deoxynojirimycin-type iminosugars is reported. Key steps in the synthetic scheme include a Dibal reduction-transimination-sodium borohydride reduction cascade of reactions on an enantiomerically pure cyanohydrin, itself prepared employing almond hydroxynitrile lyase (paHNL) as the common precursor. Ensuing ring-closing metathesis and Upjohn dihydroxylation afford the target compounds.

Lesiones retinianas producidas experimentalmente en ratas por tíner / Retinal lesions induced experimentaly in rats with thinner

Veintecuatro ratas adultas albinas inhalaron, en cámara cerrada, 1 ml. de tíner diariamente. Fueron sacrificadas por parejas a los 15, 30, 45, 60, 75 y 90 días de inhalación. Sus ojos fueron fijados en solución de formaldehído al 10%. Los cortes microscópicos estudiados mostraron al 15§. día lesiones iniciadas en los conos, células horizontales y amacrinas. En el 30§. día la capa plexiforme externa estaba adelgazada. Muchas células de todas las capas desaparecieron a los 60 días con destrucción de algunas células ganglionares, las que a los 75 días desaparecieron totalmente; sus fibras nerviosas estaban adelgazadas o desaparecidas. Al 90§. día, las capas retinianas mostraban destrucción y atrofia, incluyendo los bastones. la esclerosis agregada de capilares retinianos obstruía la luz vascular, así como los que irrigan al nervio óptico, cuyas fibras mostraban degeneración y atrofia. No había proliferación ni lesión de células neuróglicas. Estas lesiones son irreversibles