Solution structure of a DNA-binding domain from HMG1.

We have determined the tertiary structure of box 2 from hamster HMG1 using bacterial expression and 3D NMR. The all alpha-helical fold is in the form of a V-shaped arrowhead with helices along two edges and one rather flat face. This architecture is not related to any of the known DNA binding motifs. Inspection of the fold shows that the majority of conserved residue positions in the HMG box family are those involved in maintaining the tertiary structure and thus all homologous HMG boxes probably have essentially the same fold. Knowledge of the tertiary structure permits an interpretation of the mutations in HMG boxes known to abrogate DNA binding and suggests a mode of interaction with bent and 4-way junction DNA.     

AtXTH27 plays an essential role in cell wall modification during the development of tracheary elements

Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endotype hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis. Whereas recent studies have revealed temporally and spatially specific expression profiles for individual members of this family in plants, their biological roles are still to be clarified. To identify the role of each member of this gene family, we examined phenotypes of mutants in which each of the Arabidopsis XTH genes was disrupted. This was undertaken using a reverse genetic approach, and disclosed two loss-of-function mutants for the AtXTH27 gene, xth27-1 and xth27-2. These exhibited short-shaped tracheary elements in tertiary veins, and reduced the number of tertiary veins in the first leaf. In mature rosette leaves of the mutant, yellow lesion-mimic spots were also observed. Upon genetic complementation by introducing the wild-type XTH27 gene into xth27-1 mutant plants, the number of tertiary veins was restored, and the lesions disappeared completely. Extensive expression of the pXTH27::GUS fusion gene was observed in immature tracheary elements in the rosette leaves. The highest level of AtXTH27 mRNA expression in the rosette leaves was observed during leaf expansion, when the tracheary elements were elongating. These findings indicate that AtXTH27 plays an essential role during the generation of tracheary elements in the rosette leaves of Arabidopsis.     

Neutrophil CR3 expression and specific granule exocytosis are controlled by different signal transduction pathways.

Neutrophils express receptors (CR3) for the complement fragment C3bi. CR3 expression can be increased by exposure of the cells to chemotactic factors such as FMLP or to the calcium ionophore A23187. It has been suggested that CR3 moieties are stored in the membrane bounding either the secondary or the tertiary (gelatinase containing) granules. To help define the mechanisms mediating CR3 up-regulation, the effects of several inhibitors upon CR3 expression and secondary granule exocytosis were investigated. Pertussis toxin inhibited FMLP-induced (but not A23187-induced) CR3 expression and exocytosis, indicating that an early step in FMLP-induced CR3 expression is activation of a pertussis toxin-sensitive G protein. However, CR3 expression and exocytosis appeared to be controlled by separate mechanisms distal to G protein activation because 1) DBcAMP and the protein kinase inhibitor H-7 inhibited or stimulated exocytosis, respectively, without affecting CR3 expression; 2) the calmodulin (chlorpromazine and trifluoperazine) and myosin L chain kinase (ML9) inhibitors had greater effects on exocytosis than on CR3 expression; and 3) the kinetics of CR3 expression and exocytosis differed markedly. Thus, although G protein activation is a common early step in both processes, there is a bifurcation of the two processes distally. The mechanisms mediating CR3 up-regulation and tertiary granule exocytosis were also investigated. Extracellular Ca2+ was essential for tertiary granule exocytosis, but not for CR3 up-regulation. We conclude that because the mechanisms controlling CR3 up-regulation and exocytosis diverge soon after the binding of a chemotactic ligand to its receptor, that at least the bulk of increased CR3 expression is not simply a by-product of secondary and tertiary granule exocytosis but is the result of the mobilization of CR3 moieties from an intracellular pool of uncertain identity.     

Structure and receptor binding of PYY analogs

Differences in the structure of PYY and two important analogs, PYY [3-36] and [Pro34]PYY, are evaluated. Y-receptor subtype ligand binding data are used in conjunction with structural data to develop a model for receptor subtype selective agonists. For PYY it is proposed that potent binding to Y1, Y4 and Y5 receptors requires the juxtaposition of the two termini while Y2 binding only requires the C-terminal helix. Further experiments that delineate between primary and tertiary structure contributions for receptor binding and activation are required to support the hypothesis that tertiary structure is stable enough to influence the expression of PYY's bioactivity.     

Intracellular localization of a leukocyte adhesion glycoprotein family in the tertiary granules of human neutrophils

Stimulation of human neutrophils by the chemoattractant formyl-methionyl-leucyl-phenylalanine or the calcium ionophore A23187 induced increments in the cell surface expression of the alpha subunits of Mo1 (CD11b) and gp150, 95 (CD11c) and their common beta subunit (CD18). These increases showed a good correlation with the extracellular release of gelatinase, a marker for tertiary granules in human neutrophils. Conversely, the cell surface expression of the alpha subunit of the lymphocyte function-associated antigen-1 or LFA-1 (CD11a) was not altered upon cell activation. By subcellular fractionation and immunoprecipitation studies, we have found that CD11b, CD11c and CD18 molecules were mainly localized in the membranes of tertiary granules, which were resolved from the specific and azurophilic granules as well as from the plasma membrane fraction.     

Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.     

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

Glycine residues are recognized as important structural determinants in nucleotide-binding domains of many enzymes. The functional significance of seven glycine residues invariant in all 22 eNTPDase sequences was therefore examined. Glycine-to-alanine mutants of eNTPDase3 were analyzed for nucleotidase activities and tertiary and quaternary structure changes. Mutations G98A and G183A had modest effects on ATPase and ADPase activities. The G141A mutation resulted in 4- to 5-fold decreased nucleotidase activity, while the G222A mutation decreased ATPase activity 20-fold, and ADPase activity 6-fold. Unlike the other five glycine mutants, the G263A and G462A mutations caused significant loss of nucleotidase activity which was observed concomitant with lower protein expression levels, large-scale changes in tertiary and quaternary protein structure, and decreased trafficking to the plasma membrane. Thus, these data identify glycine residues that are essential for enzymatic activity and the tertiary and quaternary structure of eNTPDase3. Further, two additional conserved regions in the eNTPDases are identified, apyrase conserved regions ACR1a and ACR4a, which may be involved in phosphate binding/hydrolysis and protein folding, respectively.     

Tertiary contacts control switching of the SAM-I riboswitch

Riboswitches are non-coding RNAs that control gene expression by sensing small molecules through changes in secondary structure. While secondary structure and ligand interactions are thought to control switching, the exact mechanism of control is unknown. Using a novel two-piece assay that competes the anti-terminator against the aptamer, we directly monitor the process of switching. We find that the stabilization of key tertiary contacts controls both aptamer domain collapse and the switching of the SAM-I riboswitch from the aptamer to the expression platform conformation. Our experiments demonstrate that SAM binding induces structural alterations that indirectly stabilize the aptamer domain, preventing switching toward the expression platform conformer. These results, combined with a variety of structural probing experiments performed in this study, show that the collapse and stabilization of the aptamer domain are cooperative, relying on the sum of key tertiary contacts and the bimodal stability of the kink-turn motif for function. Here, ligand binding serves to shift the equilibrium of aptamer domain structures from a more open toward a more stable collapsed form by stabilizing tertiary interactions. Our data show that the thermodynamic landscape for riboswitch operation is finely balanced to allow large conformational rearrangements to be controlled by small molecule interactions.     

Immunolocalization of GnRHRI,gonadotropin receptors,PGR, and PGRMCI during follicular development in the rabbit ovary

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.     

Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein

Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

Intracellular location of T200 and Mo1 glycoproteins in human neutrophils

Mo1 (CD11b), a glycoprotein heterodimer that is involved in cellular adhesion processes and functions as the C3bi receptor of human myeloid cells, and T200 (CD45), a panleukocyte glycoprotein family whose function is still not well understood, increased their expression in the plasma membrane of human neutrophils after exposure to various stimuli which induce degranulation, such as formylmethionylleucylphenylalanine or calcium ionophore A23187. This increment in the expression of both molecules shows a good correlation with the release to the extracellular environment of gelatinase, a marker for an intracellular organelle named "tertiary granule" (Mollinedo, F., and Schneider, D. L. (1984) J. Biol. Chem. 259, 7143-7150). Flow cytometry studies indicate that at least 50% of the total Mo1 and T200 molecules are located in intracellular organelles. Furthermore, the subcellular distribution of Mo1 and T200 glycoproteins in resting human neutrophils was investigated by immunoprecipitation of the radiolabeled membrane proteins obtained from the distinct subcellular fractions. Both Mo1 and T200 were mainly localized in tertiary or specific intracellular granules, which were resolved from the azurophilic granules as well as from the cell membrane fraction. These findings suggest that the mobilization of intracellular Mo1 and T200 to the plasma membrane may regulate early events occurring upon neutrophil activation.     

Chemical basis of glycine riboswitch cooperativity

The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.     

Cellular activity and signaling induced by osteoprotegerin in osteoclasts: involvement of receptor activator of nuclear factor kappaB ligand and MAPK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), an inducer of osteoclastogenesis via its receptor RANK. We recently demonstrated that OPG also exerts a direct effect in osteoclasts by regulating protease expression. Herein, we showed that OPG-induced pro-matrix metalloproteinase-9 activity was abolished by ras/MAPK inhibitors in purified osteoclasts. OPG induced the phosphorylation of p38 and ERK1/2 in RAW264.7 cells. Only p38 activation was totally abolished by a blocking anti-RANKL antibody or an excess of RANKL. Surface plasmon resonance experiments revealed that RANK, RANKL and OPG are able to form a tertiary complex. These results suggested a potential formation of a tertiary complex RANK-RANKL-OPG on osteoclasts. Thus, OPG is not only a soluble decoy receptor for RANKL but must be also considered as a direct effector of osteoclast functions.     

Coupled tertiary folding and oligomerization of the T1 domain of Kv channels

Acquisition of secondary, tertiary, and quaternary structure is critical to the fabrication, assembly, and function of ion channels, yet the relationship between these biogenic events remains unclear. We now address this issue in voltage-gated K(+) channels (Kv) for the T1 domain, an N-terminal Kv recognition domain that is responsible for subfamily-specific, efficient assembly of Kv subunits. This domain forms a 4-fold symmetric tetramer. We have identified residues along the axial T1-T1 interface that are critical for tertiary and quaternary structure, shown that mutations at one end of the axial T1 interface can perturb the crosslinking of an intersubunit cysteine pair at the other end, and demonstrated that tertiary folding and tetramerization of this Kv domain are coupled. A threshold level of tertiary folding is required for monomers to oligomerize. Coupling between tertiary and quaternary structure formation may be a common feature in the biogenesis of multimeric proteins.     

The sequence determinant causing different folding behaviors of insulin and insulin-like growth factor-1

Although insulin and insulin-like growth factor-1 (IGF-1) belong to the insulin superfamily and share highly homologous sequences, similar tertiary structure, and a common ancestor molecule, amphioxus insulin-like peptide, they have different folding behaviors: IGF-1 folds into two thermodynamically stable tertiary structures (native and swap forms), while insulin folds into one unique stable structure. To further understand which part of the sequence determines their different folding behavior, based on previous reports from the laboratory, two peptide models, [B9A][1-4]porcine insulin precursor (PIP) and [B10E][1-4]PIP, were constructed. The plasmids encoding the peptides were transformed into yeast cells for expression of the peptides; the results showed that the former peptide was expressed as single component, while the latter was expressed as a mixture of two components (isomer 1 and isomer 2). The expression results together with studies of circular dichoism, disulfide rearrangement, and refolding lead us to deduce that isomer 1 corresponds to the swap form and the isomer 2 corresponds to the native form. We further demonstrate that the sequence 1-4 plus B9 of IGF-1 B-domain can make PIP fold into two structures, while sequence 1-5 of insulin B-chain can make IGF-1 fold into one unique structure. In other words, it is the IGF-1 B-domain sequence that 1-4 allows IGF-1 folding into two thermodynamically stable tertiary structures; this sequence plus its residue B9E can change PIP folding behavior from folding into one unique structure to two thermodynamically stable structures, like that of IGF-1.     

Structure and Function of Multidrug Resistance Protein 1

This review considers one of the most clinically relevant representatives of the ABC transporters–multidrug resistance protein 1 (P-glycoprotein 1 or Pgp). Data on the primary, secondary, and tertiary structure of the protein, its synthesis and degradation, and roles of its fragments in transporter activity are presented. Particular attention is given to the mechanism of functioning of Pgp. In view of the absence of a generally recognized mechanism of action of Pgp, several existing models of the protein transport cycle are discussed. Epigenetic regulation of the ABCB1 gene and modulation of Pgp expression by microRNAs are discussed.