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PubMed1981

Functional analysis of human T cell subsets defined by monoclonal antibodies. IV. Induction of suppressor cells within the OKT4+ population.

In this report, we explored the functional heterogeneity within the OKT4+ subset of human T cells. Evidence was obtained that although in vitro pokeweed mitogen-activated OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression. Despite the induction of suppressor cells after pokeweed mitogen activation, the OKT4+ population maintains its original OKT3+, OKT4+, nd OKT8- surface phenotype. The suppressor cells contained within the activated OKT4+ population were found to be radiosensitive. Importantly, the suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4+ population. Taken together, these results suggest that the OKT4+ subset of human T cells contains cells that can be activated to differentiate into suppressor cells independent of OKT8+ cells.

Antibodies↗
PubMed1989

Structural and functional analysis of insulin-like growth factors.

The insulin and insulin-like growth factor family of peptides are proteins with similar structural characteristics whose functions have diverged during evolution. Recent studies have provided a great deal of information regarding structural characteristics of these peptides, the structures of the genes that control their biosynthesis, and determination of their receptor structures. Initially these factors were believed to function as classical hormones and the endocrine regulation of their blood concentrations was studied intensively. More recently several cell types have been shown to secrete these peptides and the autocrine and paracrine regulation of insulin-like growth factor secretion by cells and tissues has received extensive characterization. Following secretion into interstitial fluids the IGF's associate with high affinity, soluble binding proteins. These proteins are capable of modulating receptor binding and growth factor biological activity. The structure of these proteins and the variables that control their secretion are currently being determined. In spite of this information significant gaps in our knowledge exist regarding the mechanisms by which these peptides stimulate replication.

Animals↗
PubMed2005

Functional analysis of Plasmodium falciparum apical membrane antigen 1 utilizing interspecies domains.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate whose function has not been unequivocally defined. Partial complementation of function can be achieved by exchanging the AMA1 of P. falciparum (PfAMA1) with that of P. chabaudi (PcAMA1). In this study, parasites expressing chimeric AMA1 proteins were created to identify domains of PfAMA1 critical in erythrocyte invasion and which are important immune targets. We report that specific chimeric AMA1 proteins containing domains I to III from PfAMA1 and PcAMA1 were able to complement PfAMA1 function in erythrocyte invasion. We demonstrate that domain III does not contain dominant epitope targets of antibodies raised against Escherichia coli expressed and refolded PfAMA1 ectodomain. Furthermore, we generated a parasite line in which the N-terminal pro region of PfAMA1 does not undergo proteolytic cleavage and show that its removal is necessary for PfAMA1 function.

Animals↗
PubMed1991

An approach to the functional analysis of lecithin-cholesterol acyltransferase. Activation by recombinant normal and mutagenized apolipoprotein AI.

Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport. The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known. Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results. This also applies to spontaneous apo AI mutants. We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction. These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter. The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity. The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).

Amino Acid Sequence↗
PubMed2002

Molecular cloning and functional analysis of a factor that binds to the proximal promoter of human angiotensinogen.

A significant association has been reported between a common variant in the angiotensinogen gene (AGT), allele T235, and essential hypertension. In subsequent work, it was found that another variant, the presence of an adenine instead of a guanine 6bp upstream from the initiation site of transcription, was in absolute linkage disequilibrium with T235. The nucleotide substitution at the -6 position affected the formation of DNA-protein complexes in gel mobility shift assays and the basal transcription of AGT in transactivation experiments. We have further examined the potential impact of this polymorphism on AGT promoter function. In ultraviolet cross-linking analysis. 150- and 75-kDa proteins bound to the AGT proximal promoter. The possible involvement of factors that bind to GC-rich domains, including Sp1, Sp3, and AP2, was not supported by gel mobility shift assays. Screening an expression library with a double-stranded DNA segment centered on -6 led to the isolation of cDNA clones encoding the YB1 protein. The specificity of the interaction of YB1 with the proximal promoter of AGT was verified by Southwestern blotting and gel mobility shift assays. In cotransfection experiments, YB1 reduced basal AGT promoter activity in a dose-dependent manner. Although these observations suggest a possible role for YB1 in modulating AGT expression, this function is likely to occur in the context of complex interactions involving other nuclear factors. The work illustrates the challenge of developing a molecular understanding of the relationship between common genetic variants and conditions that are only partly caused by them.

Angiotensinogen↗
PubMed2002

Basic functional analysis of six unknown open reading frames from Saccharomyces cerevisiae: four from chromosome VII and two from chromosome XV.

Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae from the left arms of chromosomes VII and XV were disrupted by the short-flanking homology method in the diploid strains FY1679 and CENPK2. In each case, the entire ORF, with the exception of the first nucleotide of the start codon, was eliminated and replaced by the kanMX4 cassette. Correct integration of the disrupting marker was checked by colony PCR of the geneticin (G418)-resistant transformants. Sporulation followed by tetrad dissection of the diploids revealed that none of the ORFs encoded a product essential for the viability of either yeast strain. The neutral effect of these disruptions extended to mating and sporulation, since it was possible to create homozygous diploid disruptants that were capable of sporulation. Basic phenotypic analysis was carried out on all strains by growing them on three different media at three different temperatures and revealed no significant differences between disruptants and the parental strains. A cognate clone and a kanMX4 disruption cassette were created for five of the six ORFs by gap repair with specific long-flanking homology cassettes. For experimental reasons, the cognate clone and disruption cassette corresponding to the sixth ORF (YGL161w) had to be created by PCR.

Chromosomes, Fungal↗
PubMed2004

Molecular and functional analysis of SLC25A20 mutations causing carnitine-acylcarnitine translocase deficiency.

The enzyme carnitine-acylcarnitine translocase (CACT) is involved in the transport of long-chain fatty acids into mitochondria. CACT deficiency is a life-threatening, recessively inherited disorder of lipid beta-oxidation which manifests in early infancy with hypoketotic hypoglycemia, cardiomyopathy, liver failure, and muscle weakness. We report here the clinical, biochemical, and molecular features of six CACT-deficient patients from Italy, Spain, and North America who exhibited significant clinical heterogeneity. In five patients (Patients 1, 2, 4, 5, and 6) the disease manifested in the neonatal period, while the remaining patient (Patient 3), the younger sibling of an infant who had died with clinical suspicion of fatty acid oxidation defect, has been treated since birth and was clinically asymptomatic at 4.5 years of age. Patients 1 and 4 were deceased within 6 months from the onset of this study, while the remaining four are still alive at 8, 4.5, 3.5, and 2 years, respectively. Sequence analysis of the CACT gene (SLC25A20) disclosed five novel mutations and three previously reported mutations. Three patients were homozygous for the identified mutations. Two of the novel mutations (c.718+1G>C and c.843+4_843+50del) altered the donor splice site of introns 7 and 8, respectively. The 47-nt deletion in intron 8 caused both skipping of exon 8 only and skipping of exons 6-8. Four mutations [[c.159dupT;c.163delA] ([p.Gly54Trp;p.Thr55Ala]) c.397C>T (p.Arg133Trp), c.691G>C (p.Asp231His), and c.842C>T (p.Ala281Val)] resulted in amino acid substitutions affecting evolutionarily conserved regions of the protein. Interestingly, one of these exonic mutations (p.Ala281Val) was associated with a splicing defect also characterized by skipping of exons 6-8. The deleterious effect of the p.Arg133Trp substitution was demonstrated by measuring CACT activity upon expression of the normal and the mutant protein in E. coli and functional reconstitution into liposomes. Combined analysis of clinical, biochemical, and molecular data failed to indicate a correlation between the phenotype and the genotype.

Amino Acid Sequence↗
PubMed2002

Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein.

The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

Alleles↗
PubMed2000

Functional analysis of gapped microbial genomes: amino acid metabolism of Thiobacillus ferrooxidans.

A gapped genome sequence of the biomining bacterium Thiobacillus ferrooxidans strain ATCC23270 was assembled from sheared DNA fragments (3.2-times coverage) into 1,912 contigs. A total of 2,712 potential genes (ORFs) were identified in 2.6 Mbp (megabase pairs) of Thiobacillus genomic sequence. Of these genes, 2,159 could be assigned functions by using the WIT-Pro/EMP genome analysis system, most with a high degree of certainty. Nine hundred of the genes have been assigned roles in metabolic pathways, producing an overview of cellular biosynthesis, bioenergetics, and catabolism. Sequence similarities, relative gene positions on the chromosome, and metabolic reconstruction (placement of gene products in metabolic pathways) were all used to aid gene assignments and for development of a functional overview. Amino acid biosynthesis was chosen to demonstrate the analytical capabilities of this approach. Only 10 expected enzymatic activities, of the nearly 150 involved in the biosynthesis of all 20 amino acids, are currently unassigned in the Thiobacillus genome. This result compares favorably with 10 missing genes for amino acid biosynthesis in the complete Escherichia coli genome. Gapped genome analysis can therefore give a decent picture of the central metabolism of a microorganism, equivalent to that of a complete sequence, at significantly lower cost.

Amino Acids↗
PubMed1999

Structure and functional analysis of the 26S proteasome subunits from plants.

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN 10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10delta strains which grow normally, Physcomitrella rpn10delta strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.

Arabidopsis↗
PubMed2004

Structure-function analysis of human [corrected] phosphatidylinositol transfer protein alpha bound to phosphatidylinositol.

Phosphatidylinositol transfer protein alpha (PITPalpha) selectively transports and promotes exchange of phosphatidylinositol (PI) and phosphatidylcholine (PC) between lipid bilayers. In higher eukaryotes PITPalpha is required for cellular functions such as phospholipase C-mediated signaling, regulated exocytosis, and secretory vesicle formation. We have determined the crystal structure of human PITPalpha bound to its physiological ligand, PI, at 2.95 A resolution. The structure identifies the critical side chains within the lipid-headgroup binding pocket that define the exquisite specificity for PI. Mutational analysis of the PI binding pocket is in good agreement with the structural data and allows manipulation of functional properties of PITPalpha. Surprisingly, there are no major conformational differences between PI- and PC-loaded PITPalpha, despite previous predictions. In the crystal, PITPalpha-PI is dimeric, with two identical dimers in the asymmetric unit. The dimer interface masks precisely the sequence we identify as contributing to PITPalpha membrane interaction. Our structure represents a soluble, transport-competent form of PI-loaded PITPalpha.

Amino Acid Sequence↗
PubMed2006

Cytometry on a chip: cellular phenotypic and functional analysis using grating-coupled surface plasmon resonance.

Grating-coupled surface plasmon resonance imaging (GCSPRI) is a method for the accurate assessment of both cell phenotype and function. In GCSPRI, cells and/or proteins of interest are flowed across antibodies immobilized on a gold-coated sensor chip. The surface of the chip is illuminated with monochromatic light that couples with surface plasmons in the gold. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs. Shifts in the GCSPR angle can be correlated with refractive index changes following cell or analyte capture by the immobilized antibodies. In addition, GCSPRI can image the cells as they are being captured. GCSPRI's multiplexed format allows for the parallel assessment of up to 400 individual antibody regions. In this paper, we demonstrate GCSPRI's ability to identify cells and proteins of interest and compare results to a traditional flow cytometry system. This technology represents a fast and powerful method for the simultaneous assessment of cell phenotype and function.

Animals↗
PubMed2006

New insights from the structure-function analysis of the catalytic region of human platelet phosphodiesterase 3A: a role for the unique 44-amino acid insert.

Human phosphodiesterase 3A (PDE3A) degrades cAMP, the major inhibitor of platelet function, thus potentiating platelet function. Of the 11 human PDEs, only PDE3A and 3B have 44-amino acid inserts in the catalytic domain. Their function is not clear. Incubating Sp-adenosine-3',5'-cyclic-S-(4-bromo-2,3-di-oxobutyl) monophosphorothioate (Sp-cAMPS-BDB) with PDE3A irreversibly inactivates the enzyme. High pressure liquid chromatography (HPLC) analysis of a tryptic digest yielded an octapeptide within the insert of PDE3A ((K)T(806)YNVTDDK(813)), suggesting that a substrate-binding site exists within the insert. Because Sp-cAMPS-BDB reacts with nucleophilic residues, mutants Y807A, D811A, and D812A were produced. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A. A docking model showed that Tyr(807) is 3.3 angstroms from the reactive carbon, whereas Asp(811) and Asp(812) are >15 angstroms away from Sp-cAMPS-BDB. Y807A has an altered K(m) but no change in k(cat). Activity of wild type but not Y807A is inhibited by an anti-insert antibody. These data suggest that Tyr(807) is modified by Sp-cAMPS-BDB and involved in substrate binding. Because the homologous amino acid in PDE3B is Cys(792), we prepared the mutant Y807C and found that its K(m) and k(cat) were similar to the wild type. Moreover, Sp-cAMPS-BDB irreversibly inactivates Y807C with similar kinetics to wild type, suggesting that the tyrosine may, like the cysteine, serve as a H donor. Kinetic analyses of nine additional insert mutants reveal that H782A, T810A, Y814A, and C816S exhibit an altered k(cat) but not K(m), indicating that catalysis is modulated. We document a new functional role for the insert in which substrate binding may produce a conformational change. This change would allow the substrate to bind to Tyr(807) and other amino acids in the insert to interact with residues important for catalysis in the active site cleft.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Functional analysis of the rat bile salt export pump gene promoter.

The 5' flanking region of the bile salt export pump (Bsep) gene was systematically analysed to provide the basis for understanding the mechanisms which regulate Bsep transcription. In addition substrates and drugs were investigated for their ability to alter Bsep promoter activity. Bsep promoter function was restricted to hepatocyte derived HepG2 cells. The 5' deletional analysis revealed a biphasic shape of reporter gene activities, indicating a suppressive element between nucleotides -800 and -512. Two consensus sites for the farnesoid X receptor (FXR) were located at nucleotides -473 and -64. The latter was characterized as functionally active in bile acid-mediated feed-back regulation of Bsep transcription. Bsep promoter activity was reduced by rifampin and beta-estradiol. The anti-estrogen tamoxifen stimulated promoter activity. Dexamethasone, hydrocortisone and phenobarbital had no effect on Bsep promoter activity. In conclusion, the data suggest that transcriptional regulation of the Bsep gene can be modulated by a number of endogenous compounds and xenobiotics. FXR was a major regulatory factor, mediating bile acid feed-back stimulation of Bsep transcription.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

Functional analysis of hypomethylation variants of the New Jersey serotype of vesicular stomatitis virus.

The ts mutant F1 of vesicular stomatitis virus, New Jersey serotype, directs the synthesis of undermethylated 5'-terminal cap structures in vitro. In order to determine the relationship between the ts and hypomethylation phenotypes, a spontaneous revertant rev(ts)F1 of the ts phenotype was analyzed. The revertant retained the hypomethylation phenotype. The four cap structures (GpppA, 7mGpppA, GpppAm, and 7mGpppAm) synthesized in mutant and revertant-directed reactions in the presence of low as well as high concentrations of AdoMet were resolved by HPLC. Quantitation of the data and analysis of cap substrate to product ratios revealed that despite apparent similarities between the two hypomethylation phenotypes, the functional lesions in F1 and rev(ts)F1 were different. F1 displayed an AdoMet concentration-dependent alteration in the GpppA----GpppAm reaction and an AdoMet concentration-independent alteration in the GpppA----7mGpppA reaction. In contrast, rev(ts)F1 displayed AdoMet concentration-dependent alterations in the reactions GpppA----7mGpppA and GpppAm----7mGpppAm.

Methylation↗
PubMed2000

Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target.

A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome-swelling assay was used to determine whether this protein had pore-forming capabilities. PorB had pore-forming activity and was shown to be different from MOMP porin activity.

Amino Acid Sequence↗
PubMed2006

Functional analysis of a mammalian odorant receptor subfamily.

Phylogenetic analysis groups mammalian odorant receptors into two broad classes and numerous subfamilies. These subfamilies are proposed to reflect functional organization. Testing this idea requires an assay allowing detailed functional characterization of odorant receptors. Here we show that a variety of Class I and Class II mouse odorant receptors can be functionally expressed in Xenopus laevis oocytes. Receptor constructs included the N-terminal 20 residues of human rhodopsin and were co-expressed with Galphaolf and the cystic fibrosis transmembrane regulator to allow electrophysiological measurement of receptor responses. For most mouse odorant receptors tested, these conditions were sufficient for functional expression. Co-expression of accessory proteins was required to allow functional surface expression of some mouse odorant receptors. We used this assay to examine the receptive ranges of all members of the mouse odorant receptor 42 (MOR42) subfamily. MOR42-1 responded to dicarboxylic acids, preferring a 10-12 carbon chain length. MOR42-2 responded to monocarboxylic acids (7-10 carbons). MOR42-3 responded to dicarboxylic acids (8-10 carbons) and monocarboxylic acids (10-12 carbons). Thus, the receptive range of each receptor was unique. However, overlap between the individual receptive ranges suggests that the members of this subfamily form one contiguous subfamily receptive range, suggesting that odorant receptor subfamilies do constitute functional units.

Animals↗