Cyclophilins as modulators of viral replication.

Cyclophilins are peptidyl-prolyl cis/trans isomerases important in the proper folding of certain proteins. Mounting evidence supports varied roles of cyclophilins, either positive or negative, in the life cycles of diverse viruses, but the nature and mechanisms of these roles are yet to be defined. The potential for cyclophilins to serve as a drug target for antiviral therapy is evidenced by the success of non-immunosuppressive cyclophilin inhibitors (CPIs), including Alisporivir, in clinical trials targeting hepatitis C virus infection. In addition, as cyclophilins are implicated in the predisposition to, or severity of, various diseases, the ability to specifically and effectively modulate their function will prove increasingly useful for disease intervention. In this review, we will summarize the evidence of cyclophilins as key mediators of viral infection and prospective drug targets.

Locating a lipid at the portal to the lipoxygenase active site.

Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.

Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry.

Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

Late recurrence of juvenile granulosa cell tumor of the ovary.

Juvenile granulosa cell tumor (JGCT) of the ovary, if diagnosed at an early stage, has a favorable prognosis. Recurrences are uncommon but typically occur within the first year. The patient presented here was treated with a left oophorectomy after initial presentation. Tumor recurrence in the left adnexa, diagnosed 48 months later, was treated with cytoreductive surgery followed by chemotherapy; she remains disease free 19 months after this recurrence. Late recurrences of JGCT can occur and continued close surveillance is recommended.