Purification of 26S Proteasomes and Their Subcomplexes from Plants

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type plants, and another that employs the genetic replacement of individual subunits with epitope-tagged variants combined with affinity purification. In addition to these purification protocols, we describe methods commonly used to analyze the activity and composition of the complex

Struct-NB: predicting protein-RNA binding sites using structural features

We analyse sequence and structural features of protein-RNA interfaces using RB-147, a non-redundant dataset of protein-RNA complexes extracted from the PDB. We train classifiers using machine learning algorithms to predict protein-RNA interfaces from sequence and structure-derived features of proteins. Our experiments show that Struct-NB, a Naive Bayes classifier that exploits structural features, outperforms its counterparts that use only sequence features to predict protein-RNA binding residues

An evolutionarily distinct chaperone promotes 20S proteasome alpha-ring assembly in plants

The core protease (CP) subcomplex of the 26S proteasome houses the proteolytic active sites and assumes a barrel shape comprised of four co-axially stacked heptameric rings formed by structurally related alpha- and beta-subunits. CP biogenesis typically begins with the assembly of the alpha-ring, which then provides a template for beta-subunit integration. In eukaryotes, alpha-ring assembly is partially mediated by two heterodimeric chaperones, termed Pba1-Pba2 (Add66) and Pba3-Pba4 (also known as Irc25-Poc4) in yeast. Pba1-Pba2 initially promotes orderly recruitment of the alpha-subunits through interactions between their C-terminal HbYX or HbF motifs and pockets at the alpha(5)-alpha(6) and alpha(6)-alpha(7) interfaces. Here, we identified PBAC5 as a fifth alpha-ring assembly chaperone in Arabidopsis that directly binds the Pba1 homolog PBAC1 to form a trimeric PBAC5-PBAC1-PBAC2 complex. PBAC5 harbors a HbYX motif that docks with a pocket between the alpha(4) and alpha(5) subunits during alpha-ring construction. Arabidopsis lacking PBAC5, PBAC1 and/or PBAC2 are hypersensitive to proteotoxic, salt and osmotic stresses, and display proteasome assembly defects. Remarkably, whereas PBAC5 is evolutionarily conserved among plants, sequence relatives are also dispersed within other kingdoms, including a scattered array of fungal, metazoan and oomycete species

Transcriptome and Methylome Integrative Molecular Analysis Uncovers a New Systemic Autoimmune Disease Classification

International audienc

Transcriptome and Methylome Integrative Molecular Analysis Uncovers a New Systemic Autoimmune Disease Classification

International audienc