Positively charged side chains in protein farnesyltransferase enhance catalysis by stabilizing the formation of the diphosphate leaving group

Protein farnesyltransferase (FTase) requires both Zn(2+) and Mg(2+) for efficient catalysis of the formation of a thioether bond between carbon-1 of farnesyldiphosphate (FPP) and the cysteine thiolate contained in the carboxy-terminal CaaX sequence of target proteins. Millimolar concentrations of Mg(2+) accelerate catalysis by as much as 700-fold in FTase. Although FTase lacks a typical DDXXD Mg(2+) binding site found in other enzymes that use Mg(2+) for diphosphate stabilization, D352beta in FTase has been implicated in binding Mg(2+) (Pickett et al. (2003) J. Biol. Chem. 278, 51243). Structural studies demonstrate that the diphosphate (PPi) group of FPP resides in a binding pocket made up of highly positively charged side chains, including residues R291beta and K294beta, prior to formation of an active conformation. Analysis of the Mg(2+) dependence of FTase mutants demonstrates that these positively charged residues decrease the Mg(2+) affinity up to 40-fold. In addition, these residues enhance the farnesylation rate constant by almost 80-fold in the presence of Mg(2+), indicating that these residues are not simply displaced by Mg(2+) during the reaction. Mutations at R291beta increase the pK(a) observed in the magnesium affinity, suggesting that this arginine stabilizes the deprotonated form of the PPi leaving group. Furthermore, binding and catalysis data using farnesylmonophosphate (FMP) as a substrate indicate that the side chains of R291beta and K294beta interact mainly with the beta-phosphate of FPP during the chemical reaction. These results allow refinement of the model of the Mg(2+) binding site and demonstrate that positive charge stabilizes the developing charge on the diphosphate leaving group.     

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana,Maryland, Mississippi,and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.     

Fetal bovine bone cells synthesize bone-specific matrix proteins

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

The population dynamics of pike,Esox lucius,and perch,Perca fluviatilis,in a simple predator-prey system

Synopsis The population dynamics and predator-prey relationship of pike, Esox lucius, and perch, Perca fluviatilis, were examined in simple fish communities in two adjacent shallow lakes, Lochs Kinord and Davan, Deeside, Scotland. Few perch survive to age 3 but Z is low for fish > 3 years and perch live up to 17 years. Population fecundity remained relatively high and constant in perch because of the multi-age spawning stock and the presence of older more fecund perch. Growth rates of perch in both lochs are relatively high as a consequence of low stock abundance. The N, B, and P of adult perch were unusually low. The age range of pike, and N, B, P, and growth were in the range of values reported elsewhere. There was little variation in the strength of pike year classes and the importance of cannibalism and low occurrence of alternative prey in the lochs suggest that the populations were self-regulating. Cannibalism by adults was responsible for most of mortality in perch larvae, and predation by pike and adult perch was responsible for the majority of juvenile losses. This conclusion is supported by the high biomass ratios of pike:juvenile perch of 1.0–30.8. While the number of adult fish was almost equal, the biomass of adult pike was 2–3 × that of perch in Kinord and 6 × in Davan. In L. Kinord, where year class strength was stable, high predation pressure from perch and pike reduced perch abundance rather than eliminated year classes. Perch year classes fluctuated in abundance in L. Davan and were eliminated in the first summer in two sampling years. The pike, and particularly the perch populations, have features characteristic of fish communities in unperturbed ecosystems: namely, a wide range of age classes, stability in biomass with variation dampened by longevity, and low production.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track

The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid. For example, it is not known whether NS3 can traverse covalent or physical discontinuities on the tracking strand. Here we provide evidence that NS3 translocates with a mechanism that is different from its well-studied relative, the Vaccinia helicase NPH-II. Like NPH-II, NS3 translocates along the loading strand (the strand bearing the 3'-overhang) and it fails to unwind substrates that contain nicks, or covalent discontinuities in the loading strand. However, unlike NPH-II, NS3 readily unwinds RNA duplexes that contain long stretches of polyglycol, which are moieties that bear no resemblance to nucleic acid. Whether located on the tracking strand, the top strand, or both, long polyglycol regions fail to disrupt the function of NS3. This suggests that NS3 does not require the continuous formation of specific contacts with the ribose-phosphate backbone as it translocates along an RNA duplex, which is an observation consistent with the large NS3 kinetic step size (18 base-pairs). Rather, once NS3 loads onto a substrate, the helicase can translocate along the loading strand of an RNA duplex like a monorail train following a track. Bumps in the track do not significantly disturb NS3 unwinding, but a break in the track de-rails the helicase.