Sex effects in pyelonephritis.

Urinary tract infections (UTIs) are generally considered a disease of women. However, UTIs affect females throughout the lifespan, and certain male populations (including infants and elderly men) are also susceptible. Epidemiologically, pyelonephritis is more common in women but carries increased morbidity when it does occur in men. Among children, high-grade vesicoureteral reflux is a primary risk factor for upper-tract UTI in both sexes. However, among young infants with UTI, girls are outnumbered by boys; risk factors include posterior urethral valves and lack of circumcision. Recent advances in mouse models of UTI reveal sex differences in innate responses to UTI, which vary somewhat depending on the system used. Moreover, male mice and androgenized female mice suffer worse outcomes in experimental pyelonephritis; evidence suggests that androgen exposure may suppress innate control of infection in the urinary tract, but additional androgen effects, as well as non-hormonal sex effects, may yet be specified. Among other intriguing directions, recent experiments raise the hypothesis that the postnatal testosterone surge that occurs in male infants may represent an additional factor driving the higher incidence of UTI in males under 6 months of age. Ongoing work in contemporary models will further illuminate sex- and sex-hormone-specific effects on UTI pathogenesis and immune responses.

Heterodimerization of Kinesin-2 KIF3AB Modulates Entry into the Processive Run.

Mammalian KIF3AB is an N-terminal processive kinesin-2 that is best known for its roles in intracellular transport. There has been significant interest in KIF3AB to define the key principles that underlie its processivity but also to define the mechanistic basis of its sensitivity to force. In this study, the kinetics for entry into the processive run were quantified. The results show for KIF3AB that the kinetics of microtubule association at 7 µm-1 s-1 is less than the rates observed for KIF3AA at 13 µm-1 s-1 or KIF3BB at 11.9 µm-1 s-1 ADP release after microtubule association for KIF3AB is 33 s-1 and is significantly slower than ADP release from homodimeric KIF3AA and KIF3BB, which reach 80-90 s-1 To explore the interhead communication implied by the rate differences at these first steps, we compared the kinetics of KIF3AB microtubule association followed by ADP release with the kinetics for mixtures of KIF3AA plus KIF3BB. Surprisingly, the kinetics of KIF3AB are not equivalent to any of the mixtures of KIF3AA + KIF3BB. In fact, the transients for each of the mixtures overlay the transients for KIF3AA and KIF3BB. These results reveal that intermolecular communication within the KIF3AB heterodimer modulates entry into the processive run, and the results suggest that it is the high rate of microtubule association that drives rebinding to the microtubule after force-dependent motor detachment.

Kinesin-2 KIF3AB exhibits novel ATPase characteristics.

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 µm(-1) s(-1), followed by rate-limiting ADP release at 12.8 s(-1). ATP binding at 7.5 µm(-1) s(-1) was followed by an ATP-promoted isomerization at 84 s(-1) to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s(-1). ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s(-1). The dissociation step showed an apparent affinity for ATP that was very weak (K½,ATP at 133 µm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 µm(-1) s(-1), which is inconsistent with fast ATP binding at 7.5 µm(-1) s(-1) and a Kd ,ATP at 6.1 µm. These results suggest that ATP binding per se cannot account for the apparent weak K½,ATP at 133 µm. The steady-state ATPase Km ,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.

Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi.

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 Å and a heavy atom root mean square deviation of 0.93 Å. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra ß-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.

1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein.

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.