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81.
M Kanda A Jablonka-Shariff A Sato M R Pixley E Bos T Hiro'oka D Ben-Menahem I Boime 《Molecular endocrinology (Baltimore, Md.)》1999,13(11):1873-1881
The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles. 相似文献
82.
83.
Sabirova AR Rudakova NL Balaban NP Ilyinskaya ON Demidyuk IV Kostrov SV Rudenskaya GN Sharipova MR 《FEBS letters》2010,584(21):4419-4425
The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes. 相似文献
84.
Cherepanova O Orlova A Galkin VE van der Ven PF Fürst DO Jin JP Egelman EH 《Journal of molecular biology》2006,356(3):714-723
Xin and nebulette are striated muscle-specific actin-binding proteins that both contain multiple actin-binding repeats. The nature of these repeats is different: nebulette has nebulin-like repeats, while Xin contains its own unique repeats. However, the suggestion was made from biochemical data that the Xin-repeats may bind to multiple sites on the actin molecule as was found for nebulin. We have used electron microscopy and the iterative helical real space reconstruction to visualize complexes of F-actin with Xin fragments containing either three or six Xin-repeats, and with the CN5-nebulette fragment, containing five nebulin-like repeats. Our results indicate that Xin and nebulette fragments bind to F-actin in a similar manner and in two distinct modes: in one mode actin subdomain 1 is bound, while in the second mode the binding bridges between a different site on actin subdomains 1/2 of one protomer and subdomains 3/4 of an adjacent actin protomer. Taken together with published data about nebulin, tropomyosin and ADF/cofilin, our results suggest that the ability to bind in multiple modes to the actin protomer is a general property of many actin-binding proteins. 相似文献
85.
Many actin-binding proteins have been observed to have a modular architecture. One of the most abundant modules is the calponin-homology (CH) domain, found as tandem repeats in proteins that cross-link actin filaments (such as fimbrin, spectrin and alpha-actinin) or link the actin cytoskeleton to intermediate filaments (such as plectin). In proteins such as the eponymous calponin, IQGAP1, and Scp1, a single CH-domain exists, but there has been some controversy over whether this domain binds to actin filaments. A previous three-dimensional reconstruction of the calponin-F-actin complex has led to the conclusion that the visualized portion of calponin bound to actin belongs to its amino-terminal homology (CH) domain. We show, using a calponin fragment lacking the CH-domain, that this domain is not bound to F-actin, and cannot be positioning calponin on F-actin as hypothesized. Further, using classification methods, we show a multiplicity in cooperative modes of binding of calponin to F-actin, similar to what has been observed for other actin-binding proteins such as tropomyosin and cofilin. Our results suggest that the form and function of the structurally conserved CH-domain found in many other actin-binding proteins have diverged. This has broad implications for inferring function from the presence of structurally conserved domains. 相似文献
86.
Lavigne LM Albina JE Reichner JS 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(12):8667-8675
Candida albicans is a common cause of nosocomial infections whose virulence depends on the reversible switch from blastoconidia to hyphal forms. Neutrophils (or polymorphonuclear leukocytes (PMNs)) readily clear blastoconidia by phagocytosis, but filaments are too long to be ingested. Mechanisms regulating immune recognition and response to filamentous fungal pathogens are not well understood, although known risk factors for developing life-threatening infections are neutropenia or defects in the NADPH oxidase system. We show human PMNs generate a respiratory burst response to unopsonized hyphae. Ab specific for beta-glucan, a major component of yeast cell walls, blocks this response, establishing beta-glucan as a key molecular pattern recognized by PMNs in response to C. albicans. This study also elucidates recognition and signaling mechanisms used by PMNs in response to beta-glucan under conditions where phagocytosis cannot occur. Human PMNs adhered to immobilized beta-glucan and released an efficient plasma membrane respiratory burst. Ab blockade of the integrin complement receptor 3 (CD11b/CD18) significantly inhibited both of these functions. Furthermore, we show a role for p38 MAPK and actin but not protein kinase C zeta in generating the respiratory burst to beta-glucan. Taken together, results show that beta-glucan in C. albicans hyphae is accessible to PMNs and sufficient to support an innate immune response. 相似文献
87.
Jablonka-Shariff A Kumar TR Eklund J Comstock A Boime I 《Molecular endocrinology (Baltimore, Md.)》2006,20(6):1437-1446
The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition. 相似文献
88.
A carboxyl-terminal sequence in the lutropin beta subunit contributes to the sorting of lutropin to the regulated pathway 总被引:1,自引:0,他引:1
Jablonka-Shariff A Pearl CA Comstock A Boime I 《The Journal of biological chemistry》2008,283(17):11485-11492
Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid-estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identicalalpha subunit, and thus thebeta subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHbeta subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHbeta subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHDeltaT (LHbeta subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH(3) cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHDeltaT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHDeltaT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHDeltaT compared with LH, consistent with increased constitutive secretion of LHDeltaT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHbeta subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway. 相似文献
89.
Albina Jablonka-Shariff Vicenta Garcia-Campayo Irving Boime 《The Journal of biological chemistry》2002,277(2):879-882
One of the fundamental differences among mammals is the mechanism of maintaining the corpus luteum of pregnancy. Placentation in primates is associated with the production of the glycoprotein hormone chorionic gonadotropin (CG), which is secreted into the maternal serum and stimulates progesterone synthesis from the corpus luteum, which is essential for early development of the embryo. CG together with the pituitary hormones lutropin (LH), follitropin, and thyrotropin constitute the family of glycoprotein hormones comprised of a common alpha subunit and a hormone-specific beta subunit. The LHbeta and CGbeta subunits share 85% amino acid sequence identity, and functionally LH and CG are interchangeable. CGbeta evolved by a recent gene duplication event from the LHbeta locus, and despite the close relationship between them, their modes of secretion are quite different. CG release from the placenta is apically directed, whereas LH is released from the basal side of the cell, and the determinant(s) for this redirected trafficking are unknown. Here, using the polarized Madin-Darby canine kidney (MDCK) cell line, we provide evidence for the molecular basis of the different secretory patterns of LH and CG in vivo. The apical targeting of CG is programmed by a carboxyl-terminal sequence, which encodes a novel sorting signal. It is also apparent that the presence of the O-linked oligosaccharides in the CTP sequence contributes to this apical routing. The CTP, which is absent in LH, redirects CG to the maternal serum and permits the unique arrangement for primate placentation. Our data also show that the MDCK cells can distinguish the different secretory pathways for the gonadotropins and will be a valuable model for elucidating the determinants associated with the unique sorting of these functionally related hormones. 相似文献
90.
Dott. Albina Messeri 《Plant biosystems》2013,147(1):78-127
Bupleurum gracile (M. Bieb.) DC. var. rollii Montel. ( ≡ Bupleurum rollii [Montel.] Moraldo) is lectotypified on a herbarium specimen kept in RO, revising the previous typification by S. Snogerup and B. Snogerup. An isolectotypes and 14 syntypes are also selected. Infraspecific variability and comparison with related Eastern species B. asperuloides Heldr. B. uechtritzianum S. Stoyanov and B. marschallianum C. A. Mey plus B. gracile D'Urv. are studied using morphometric and anatomical data. Notes on the ecology and distribution of the Italian endemic taxon are also provided. 相似文献