Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for subtilases in plant biology. We report the first structure of a plant subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal sevenstranded jelly-roll fibronectin (Fn) III-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although Ca 2؉ -binding sites are conserved and critical for stability in other subtilases, SBT3 was found to be Ca 2؉ -free and its thermo stability is Ca 2؉ -independent.calcium ͉ proprotein convertase ͉ protease-associated domain ͉ subtilisin ͉ thermostability S ubtilases constitute the S8 family in clan SB of serine proteases (http://merops.sanger.ac.uk). They are characterized by a catalytic triad of Asp, His, and Ser residues in an arrangement shared with subtilisins from Bacillus species (1). The first eukaryotic subtilase to be identified was kexin. It is involved in the maturation of ␣-mating factor and killer toxin from their respective precursor proteins in yeast (2). Nine subtilisin-like endopeptidases have since been discovered in mammals, seven of which are related to kexin and also involved in the highly specific processing of precursor proteins. Their substrates include polypeptide hormone precursors, growth factors, receptors, enzymes, and viral surface glycoproteins, which are typically cleaved on the carboxyl side of paired basic residues (3). The remaining two subtilases, PCSK9 and S1P, belong to the proteinase K and pyrolysin subfamilies of subtilases (3). The discovery of mammalian proprotein convertases (PCs), characterized by their exquisite substrate specificity compared with bacterial subtilisins, further stimulated interest in this class of serine proteases.Plants appear to lack kexin-related PCs but they possess a largely expanded pyrolysin family with 56 genes identified in Arabidopsis thaliana (4). They have been implicated in general protein turnover (5), the regulation of plant development (6), biotic and abiotic stress responses (7,8), and the processing of precursors of peptide growth factors in plants (9). It therefore seems that the majority of plant subtilases assumed plant-specific functions in the course of evolution. With the physiological roles of plant subtilases beginning to emerge, it will now be interesting to see whether or not the adoption of specific roles in plant physiology is reflected in unique structural or biochemical features that distinguish subtilases in plants from those in other organisms.To address this question we recently purified and cha...

Background: Protein quality control proteases degrade damaged proteins and protein fragments. Results:The human serine protease HTRA1 degrades tau aggregates and is induced by its substrates. Conclusion: A member of the widely conserved HtrA family is involved in protein quality control in mammalian cells. Significance: HTRA1 might function as a tau protease in vivo.

Channeling of misfolded proteins into repair, assembly or degradation pathways is often mediated by complex and multifunctional cellular factors. Despite detailed structural information, the underlying regulatory mechanisms governing these factors are not well understood. The extracytoplasmic heat-shock factor DegP (HtrA) is a well-suited model for addressing mechanistic issues, as it is regulated by the common mechanisms of allostery and activation by oligomerization. Site-directed mutagenesis combined with refolding and oligomerization studies of chemically denatured DegP revealed how substrates trigger the conversion of the resting conformation into the active conformation. Binding of specific peptides to PDZ domain-1 causes a local rearrangement that is allosterically transmitted to the substrate-binding pocket of the protease domain. This activated state readily assembles into larger oligomeric particles, thus stabilizing the catalytically active form and providing a degradation cavity for protein substrates. The implications of these data for the mechanism of protein quality control are discussed.

Allostery is a basic principle of control of enzymatic activities based on the interaction of a protein or small molecule at a site distinct from an enzyme's active center. Allosteric modulators represent an alternative approach to the design and synthesis of small-molecule activators or inhibitors of proteases and are therefore of wide interest for medicinal chemistry. The structural bases of some proteinaceous and small-molecule allosteric protease regulators have already been elucidated, indicating a general mechanism that might be exploitable for future rational design of small-molecule effectors.