A Completely De Novo ATPase from Combinatorial Protein Design.

Our understanding of biological chemistry is shaped by the observation that all life comes from other life-as Pasteur put it, omne vivum ex vivo. A key step in expanding our biochemical vocabulary is to recapitulate biogenic catalysis using non-natural sequences that did not arise from common ancestry. Here we describe an enzyme designed completely de novo that hydrolyzes ATP. This protein was designed to lack ß-sheet structure and is competitively inhibited by magnesium, two traits that are unlike natural ATPases.

Chemical synthesis of the ATAD2 bromodomain.

Due to the emerging importance of the bromodomain binding region in the study of epigenetic effectors and the vast implications for a wide variety of human disease, the bromodomain region of human ATPase family AAA+ (ATPases associated with diverse cellular activities) domain-containing protein 2 (ATAD2) was targeted for chemical synthesis. The ATAD2 bromodomain (130 aa) was divided into five strategic fragments to be assembled using native chemical ligation with a focus on maximal convergency and efficiency. The fragments were assembled with one cysteine and three thioleucine ligations, unveiling the native alanine and leucine amino acids at the ligation points following metal-free dethiylation. Synthetic highlights of the study are a photolabile dimethoxynitrobenzyl-protected glutamic acid side chain used to impede hydrolysis of the C-terminal Glu-thioester, a thiazolidine-protected thioleucine, and an efficient assembly of three fragments in a single reaction vessel with dual-mode kinetic-standard chemical ligation. With a focus on material throughput and convergency, the five peptide fragments were assembled into the native ATAD2 bromodomain region with a total of three HPLC events in 8% overall yield from the fragments.

Synthetic salusins as cardiac depressors in rat.

Using bioinformatic analyses of full-length, enriched human cDNA libraries, we recently identified salusins, multifunctional related peptides ubiquitously expressed in major human tissues. Salusins cause transient and profound hypotension when injected intravenously to rats, the hypotensive effect of salusin-beta being especially striking. However, the mechanisms of this hypotensive action remain elusive. To determine whether salusins modulate cardiac function in rats, we studied serial changes of systemic hemodynamics and functions of isolated perfused working and nonworking hearts before and after salusin administration. Intravenous salusin-beta administration to intact anesthetized rats caused a temporary rapid, profound decrease in aortic blood flow concomitantly with hypotension and bradycardia without affecting systemic vascular resistance. Salusin-beta-induced hypotension and bradycardia were completely blocked by pretreatment with atropine, a muscarinic receptor antagonist, but not by propranolol. In isolated perfused working rat hearts, salusin-beta significantly decreased cardiac output, aortic flow, and stroke work. However, it did not affect coronary flow in isolated working and nonworking hearts. Our results indicate that salusins induce potent hypotension via negative inotropic and chronotropic actions. Salusin-beta promotes its actions by facilitating vagal outflows to the heart, whereas the negative inotropism of salusin-beta is also mediated via a direct myotropic effect.