We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)–dependent signal transduction, which is important in many cellular processes. Signals received by the G protein–coupled receptors are conveyed to ACs through G proteins to modulate the levels of cellular cyclic adenosine monophosphate (cAMP). Here, we describe the cryo–electron microscopy structure of the bovine membrane AC9 bound to an activated G protein αs subunit at 3.4-angstrom resolution. The structure reveals the organization of the membrane domain and helical domain that spans between the membrane and catalytic domains of AC9. The carboxyl-terminal extension of the catalytic domain occludes both the catalytic and the allosteric sites of AC9, inducing a conformation distinct from the substrate- and activator-bound state, suggesting a regulatory role in cAMP production.

Adenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to the pro-apoptotic factor Apaf-1 and the activation of the caspase-9 and -3 pathways. In this study it is shown, by means of several assays on whole cells and isolated mitochondria, that 2-chloro-2′-deoxyadenosine (2CdA) and 2-choloro-2′-ara-fluorodeoxyadenosine (CaFdA) disrupt the integrity of mitochondria from primary chronic lymphocytic leukemia (B-CLL) cells. The nucleoside-induced damage leads to the release of the pro-apoptotic mitochondrial proteins cytochrome c and apoptosis-inducing factor. The other adenine deoxynucleosides tested displayed comparable DNA-damaging potency but did not affect mitochondrial function. Interference with mitochondrial integrity, thus, may be a factor in the potent cytotoxic effects of 2CdA and CaFdA toward nondividing lymphocytes.

Adenine deoxynucleosides, such as 2-chloro-2-deoxyadenosine (2CdA) induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. However, it has remained puzzling why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis. The present experiments demonstrate that the 5-triphosphate metabolite of 2CdA (2CdA-5-triphosphate), similar to dATP, can cooperate with cytochrome c and Apaf-1 to activate caspase-3 in a cell free system. Chronic lymphocytic leukemia cells and normal peripheral blood lymphocytes expressed both caspase-3 and apoptotic protease activating factor 1. Incubation of the lymphocytes with 2CdA induced caspase-3 activation prior to DNA degradation and cell death. Stimulation of the caspase proteolytic cascade by 2CdA-5-triphosphate, in the context of DNA strand break formation, may provide an explanation for the potent cytotoxic effects of 2CdA toward nondividing lymphocytes.The effectiveness of cancer chemotherapy often depends upon the induction of apoptosis in malignant cells. Among antimetabolites, the 2Ј-deoxyadenosine congeners 2-chloro-2Ј-deoxyadenosine (2CdA, cladribine) and 9-␤-D-arabinofuranosyl-2-fluoroadenine (fludarabine) have the ability to induce apoptosis in nondividing lymphocytes, at concentrations that spare other cell types (1). For this reason, the deoxyadenosine analogs have achieved an important place in the treatment of indolent lymphoid malignancies, including hairy cell leukemia, chronic lymphocytic leukemia (CLL), and low grade lymphoma (2, 3).The cytotoxicity of 2CdA depends mainly upon the selective and progressive accumulation of its 5Ј-triphosphate metabolite (2CdATP) in lymphocytes that have a high ratio of deoxycytidine kinase (EC 2.7.1.74) to cytosolic 5Ј-nucleotidase (EC 3.1.3.5), compared with other cell types (1, 4). However, why 2CdATP triggers apoptosis in non-dividing cells is unclear.Various stimuli of apoptosis lead to the activation in the cytoplasm of cysteine proteases with specificity for aspartic acid residues, referred to as caspases. The activated caspases can cleave structural proteins and enzymes necessary for the survival of both proliferating and resting cells (reviewed in refs. 5-7). In addition, caspases have been shown to activate the endonuclease responsible for the internucleosomal cleavage of genomic DNA, a hallmark of apoptosis (8,9).One important component of the caspase cascade is caspase-3, which is activated by two sequential proteolytic events that cleave the 32-kDa precursor at aspartic acid residues to generate an active heterodimer of 20-and 12-kDa subunits (10). The activation can either be autocatalytic, or occur via a caspase cascade, similar to the serine protease cascade in the blood clotting process (7). In susceptible cells, caspase activation might amplify preexisting but sublethal apoptotic signals, leading to rapid and irreversible proteolysis.Recently, Wang and coworkers established a cell free system in ...