A rare variant in human fibroblast activation protein associated with ER stress,loss of enzymatic function and loss of cell surface localisation

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells.     

Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila

Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.     

Activity and Biospecificity of Proteolyzed Forms and Dimeric Combinations of Recombinant Human and Murine Nerve Growth Factor

Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland-derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed-phase HPLC (RP-HPLC), and high-performance ion-exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120-residue form of rhNGF produced a species of 109 residues (10-118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N-terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N-terminal sequence, PAGE, and RP-HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma-12 cell line neurite extension assay. The 118-residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal-modified molecules presented activity reduced by 50- to 100-fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.     

Determinants of L-aspartate and IMP recognition in Escherichia coli adenylosuccinate synthetase.

Adenylosuccinate synthetase governs the first committed step in the de novo synthesis of AMP. Mutations of conserved residues in the synthetase from Escherichia coli reveal significant roles for Val(273) and Thr(300) in the recognition of l-aspartate, even though these residues do not or cannot hydrogen bond with the substrate. The mutation of Thr(300) to alanine increases the K(m) for l-aspartate by 30-fold. In contrast, its mutation to valine causes no more than a 4-fold increase in the K(m) for l-aspartate, while increasing k(cat) by 3-fold. Mutations of Val(273) to alanine, threonine, or asparagine increase the K(m) for l-aspartate from 15- to 40-fold, and concomitantly decrease the K(i) for dicarboxylate analogues of l-aspartate by up to 40-fold. The above perturbations are comparable with those resulting from the elimination of a hydrogen bond between the enzyme and substrate: alanine mutations of Thr(128) and Thr(129) increase the K(m) for IMP by up to 30-fold and the alanine mutation of Thr(301) abolishes catalysis supported by l-aspartate, but has no effect on catalysis supported by hydroxylamine. Structure-based mechanisms, by which the above residues influence substrate recognition, are presented.     

3-dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.     

Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.

The lentiviruses of sheep, goats, and horses cause chronic multiorgan disease in which macrophages are highly permissive for viral replication. Monocytes, which mature into macrophages, are thought to be latently infected with lentivirus, but the extent to which other leukocytes are infected is unknown. Dendritic cells have not been studied separately from monocytes and T-cell subsets have not been examined in previous attempts to identify infected cells in peripheral blood mononuclear cells (PBMC). We found no evidence of T-cell tropism using an animal-passaged, pathogenic ovine lentivirus. Phytohemagglutinin-stimulated infectious PBMC produced 20-fold less virus than differentiated macrophages, and cocultivation of infectious PBMC with fresh, uninfected phytohemagglutinin blasts did not facilitate virus replication. Furthermore, central lymph cells, the best in vivo source of purified lymphocytes, lacked virus and did not yield virus upon in vitro cultivation. In contrast, cultivated blood-derived macrophages were highly permissive for viral replication. To identify the latently infected PBMC, PBMC from infected sheep were selectively depleted of monocytes and B cells by passage over nylon wool and then of nonadherent cells bearing CD4, CD8, T19, gamma delta T-cell receptor, CD45RA, or major histocompatibility complex class II antigens by panning. Removal of adherent monocytes and B cells or of adherent cells and the three major T-cell subsets (CD4+, CD8+, T19+) did not decrease the infectivity of PBMC. The richest sources of infected cells in fresh PBMC were CD45RA+ and major histocompatibility complex class II+ nonadherent cells, which are three characteristics of dendritic cells. Thus, the dendritic cell, and not the monocyte or the CD4+ cell, is probably the predominant infected cell type in blood.     

A bioinformatic strategy for the detection, classification and analysis of bacterial autotransporters

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.     

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.     

The K = 2 conundrum

Assessments of population genetic structure have become an increasing focus as they can provide valuable insight into patterns of migration and gene flow. structure , the most highly cited of several clustering‐based methods, was developed to provide robust estimates without the need for populations to be determined a priori. structure introduces the problem of selecting the optimal number of clusters, and as a result, the ΔK method was proposed to assist in the identification of the “true” number of clusters. In our review of 1,264 studies using structure to explore population subdivision, studies that used ΔK were more likely to identify K = 2 (54%, 443/822) than studies that did not use ΔK (21%, 82/386). A troubling finding was that very few studies performed the hierarchical analysis recommended by the authors of both ΔK and structure to fully explore population subdivision. Furthermore, extensions of earlier simulations indicate that, with a representative number of markers, ΔK frequently identifies K = 2 as the top level of hierarchical structure, even when more subpopulations are present. This review suggests that many studies may have been over‐ or underestimating population genetic structure; both scenarios have serious consequences, particularly with respect to conservation and management. We recommend publication standards for population structure results so that readers can assess the implications of the results given their own understanding of the species biology.