Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.     

Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases delta-selinene synthase and gamma-humulene synthase

Two recombinant sesquiterpene synthases from grand fir, delta-selinene synthase and gamma-humulene synthase, each produce more than 30 sesquiterpene olefins from the acyclic precursor farnesyl diphosphate. These enzymes contain a pair of DDxxD motifs, on opposite lips of the presumptive active site, which are thought to be involved in substrate binding and could promote multiple orientations of the substrate alkyl chain from which multiple families of cyclic olefins could derive. Mutagenesis of the first aspartate of either DDxxD motif resulted in depressed k(cat), with lesser effect on K(m), for delta-selinene synthase and afforded a much simpler product spectrum composed largely of monocyclic olefins. Identical alterations in gamma-humulene synthase produced similar kinetic effects with a simplified product spectrum of mostly acyclic and monocyclic olefins. Although impaired in product diversity, none of the mutant synthases lost entirely the capacity to generate complex structures. These results confirm the catalytic significance of the DDxxD motifs and imply that they also influence permitted modes of cyclization. Deletion of an N-terminal arginine pair in delta-selinene synthase (an element potentially involved in substrate isomerization) altered kinetics without substantially altering product outcome. Finally, mutation of an active-site tyrosine residue thought to play a role in proton exchange had little influence; however, substitution of a nearby active site aspartate dramatically altered kinetics and product outcome.     

Tyrosylprotein sulfotransferase inhibitors generated by combinatorial target-guided ligand assembly

Tyrosylprotein sulfotransferases (TPSTs) catalyze the sulfation of tyrosine residues within secreted and membrane-bound proteins. The modification modulates protein-protein interactions in the extracellular environment. Here we use combinatorial target-guided ligand assembly to discover the first known inhibitors of human TPST-2.     

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.     

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.     

pH reduction as a trigger for dissociation of herpes simplex virus type 1 scaffolds

Assembly of the infectious herpes simplex virus type 1 virion is a complex, multistage process that begins with the production of a procapsid, which is formed by the condensation of capsid shell proteins around an internal scaffold fashioned from multiple copies of the scaffolding protein, pre-VP22a. The ability of pre-VP22a to interact with itself is an essential feature of this process. However, this self-interaction must subsequently be reversed to allow the scaffolding proteins to exit from the capsid to make room for the viral genome to be packaged. The nature of the process by which dissociation of the scaffold is accomplished is unknown. Therefore, to investigate this process, the properties of isolated scaffold particles were investigated. Electron microscopy and gradient sedimentation studies showed that the particles could be dissociated by low concentrations of chaotropic agents and by moderate reductions in pH (from 7.2 to 5.5). Fluorescence spectroscopy and circular dichroism analyses revealed that there was relatively little change in tertiary and secondary structures under these conditions, indicating that major structural transformations are not required for the dissociation process. We suggest the possibility that dissociation of the scaffold may be triggered by a reduction in pH brought about by the entry of the viral DNA into the capsid.     

The gene for juvenile hyaline fibromatosis maps to chromosome 4q21

Juvenile hyaline fibromatosis (JHF) is an autosomal recessive condition characterized by multiple subcutaneous nodular tumors, gingival fibromatosis, flexion contractures of the joints, and an accumulation of hyaline in the dermis. We performed a genomewide linkage search in two families with JHF from the same region of the Indian state of Gujarat and identified a region of homozygosity on chromosome 4q21. Dense microsatellite analyses within this interval in five families with JHF who were from diverse origins demonstrate that all are compatible with linkage to chromosome 4q21 (multipoint LOD score 5.5). Meiotic recombinants place the gene for JHF within a 7-cM interval bounded by D4S2393 and D4S395.     

Free tissue coverage of chronic traumatic wounds of the lower leg

Thirty-eight consecutive patients who underwent 42 free flaps for chronic wounds of the lower leg were identified over an 11-year period. All wounds were open for a minimum of 1 month (mean, 40 months; median, 8 months; range, 1 month to 30 years). The average age was 37 years (range, 7 to 68 years), there were 31 male patients and seven female patients, and the average follow-up time was 30 months (range, 12 to 72 months). The original injury was an open fracture in 28 patients, wound dehiscence after open reduction and internal fixation of a closed fracture in nine patients, and a shrapnel wound in one patient. A total of 23 patients had osteomyelitis, which was classified as local (involving less than 50 percent of the bone diameter) in 15 patients and as diffuse (involving greater than 50 percent of the bone diameter or infected nonunion) in eight patients. The wounds were treated with sequential debridement, antibiotics, and flap coverage. Ancillary procedures included antibiotic beads in 18 patients, saucerization in 16, Ilizarov bone transport in three, calcanectomy in two, and fibular resection and ankle fusion in one. Thirty-four of 42 flaps survived, four having undergone a repeat free flap. There were three failures out of 25 flaps (12 percent) among those with a normal angiogram and five failures out of 15 flaps (33 percent) among those with an abnormal angiogram (p > 0.05). The failure rate of those with osteomyelitis was six of 26 (23 percent) versus two of 26 (13 percent) for those without osteomyelitis (p > 0.05). Successful reconstruction (bone healed, patient ambulatory and infection-free) was achieved in 33 of 38 patients (87 percent). The failure of reconstruction for those patients with osteomyelitis was four of 23 (22 percent) versus one of 15 (7 percent) for others (p > 0.05). The failure rate of flaps in patients with diffuse osteomyelitis was three of eight (38 percent) versus two of 30 for others (7 percent, p = 0.053). The presence of diffuse osteomyelitis was associated with a lower rate of successful limb reconstruction. An abnormal angiogram and the presence of osteomyelitis both were associated with a lower rate of successful limb reconstruction, but this was not significant, probably because of the small size of the cohort.     

Thermoregulation of weaned northern elephant seal (Mirounga angustirostris) pups in air and water

Fasting weaned northern elephant seal pups (Mirounga angustirostris) experience diverse environmental conditions on land and in water on a daily basis. Each environment undoubtedly induces distinct energetic costs that may vary for pups of differing body condition. To determine the energetic costs associated with different environmental conditions and whether costs vary between individuals, body mass, surface area, volume, body composition, resting metabolic rate, and core body temperature were determined for 17 weaned northern elephant seal pups from A?o Nuevo, California. Metabolic rate and body temperature were measured for pups resting in air (20.9 degrees +/-0.8 degrees C), cold water (3.8 degrees+/-0.4 degrees ;C), and warm water (14.5 degrees+/-0.2 degrees C). Resting metabolic rate increased with body mass (range: 62.0-108.0 kg) and was also correlated with lean mass and lipid mass. Metabolic rates ranged from 293.6 to 512.7 mL O(2) min(-1) and were lowest for pups resting in cold water. Thermal conductance, calculated from metabolic rate and core body temperature, ranged from 3.1 to 15.2 W degrees C(-1), with the highest values in air and the lowest values in cold water. Metabolic responses to the three environmental conditions did not differ with individual variation in body condition. For all elephant seal pups, a consequence of high lipid content is that thermoregulatory costs are greatest on land and lowest in cold water, a pattern that contrasts markedly with terrestrial mammals.