Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.epigenetics | protein-protein complex | A9145C P osttranslational methylation of lysine and arginine residues by protein lysine methyltransferases and protein arginine methyltransferases (PRMTs) alters the activity and interactions of substrate proteins, with crucial consequences to diverse cellular functions (1-3). Histone methylation is an epigenetic mark that plays a vital role in normal cell function, and whose dysregulation is associated with several diseases (4).The PRMT family of methyltransferases belongs to the largest class (class I) of S-adenosylmethionine (AdoMet)-dependent methyltransferase enzymes, responsible for the transfer of a methyl group from AdoMet to the arginine side-chains of histones and other proteins. PRMTs are further subdivided into type I, type II, type III, and type IV enzymes based on their patterns of arginine methylation. Eleven human PRMTs have been identified to date (5), and they all methylate the terminal guanidino nitrogen atoms of arginine residues. Type I PRMT enzymes (PRMT1, -2, -3, -4, -6, and -8) generate ω-NG-monomethyl and ω-NG,NG-asymmetric di-methyl arginines, whereas PRMT5 is a type II PRMT that catalyzes the formation of ω-NG-monomethyl and ω-NG,N′G-symmetric di-methyl arginine residues. PRMT7 was initially thought to have type II activity, but recent evidence suggests that it may be a type III enzyme that is only able to monomethylate substrates to form ω-NG-monomethyl arginine (6). A type IV enzyme that catalyses the formation of δ-N-methyl arginine has been identified in yeast (7). All PRMTs share the highly conserved methyltransferase catalytic domain, and several PRMTs contain additional domains that modulate their activity and specificity. PRMT2, PRMT3, and PRMT9 contain SH3, zinc finger, and TRP2 domains, respectively, and PRMT5 contains a largely uncharacterized N-terminal region.In contrast to type I PRMTs, PRMT5 functions as part of various high molecular weight protein complexes that invariably contain the WD-repe...

The results indicate that opalescent appearance is not due to self-association but is a simple consequence of Rayleigh scatter. Opalescent appearance did not result in physical instability.

During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH. The presence of residual levels of protease activity in purified protein batches affected the inherent activity of the product when stored as a solution. To develop a robust purification strategy to minimize this undesirable impact, identification and characterization of this protease was essential to ultimately ensure that a solution formulation was stable for many years. A protease was isolated from CHO cell-free medium (CFM) using a combination of immobilized pepstatin-A agarose chromatography and size exclusion chromatography (SEC). The isolated protease has significant proteolytic activity at pH ∼ 3 to neutral pH and was identified as cathepsin D by mass spectrometry. Analytical SEC, chip-based capillary gel electrophoresis, imaged capillary isoelectric focusing (cIEF), and circular dichroism (CD) spectropolarimetry analyses were performed for additional characterization of the protease. The identification and characterization of this protease enabled the development of a robust purification process by implementation of a controlled temperature inactivation unit operation (heat inactivation) that enabled essentially complete inactivation of the protease, resulting in the production of a stable drug product that had not been possible using column chromatography alone. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:120-129, 2018.

No abstract

Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1). The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.