Biomedical subjects
Publications and source records attributed to J Gergely.
In the absence of phosphorylated Gab2, immature B cells are unable to maintain a prolonged signal transduction, which is necessary for the transcriptional regulation and activation of the cells. Thus, the lack of Gab2 phosphorylation may drive immature B cells to apoptosis.
We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnC's N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.
The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcgamma receptor (FcgammaRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgammaRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgammaRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgammaRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgammaRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to the phosphorylated FcgammaRIIb. SHP-2, activated upon the binding to FcgammaRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgammaRIIb co-aggregated cells.
Skeletal muscle troponin C (TnC) adopts an extended conformation when crystallized alone and a compact one when crystallized with an N-terminal troponin I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's inhibitory region between TnC and actin [Tripet et al. (1997) J. Mol. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluorescence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of photo-cross-linking in TnC were determined by microsequencing and mass spectrometry following enzymatic digestions. Our results show that in the troponin complex neither the distance between TnI residue 6 and TnC residue 89 nor the photo-cross-linking site in TnC, Ser133, changes with Ca(2+), in support of the notion that this region plays mainly a structural rather than a regulatory role. The distances to residues 12 and 41 in TnC's N-domain are both considerably longer than those predicted by the crystal structure of TnC.TnI(1-47), supporting an extended rather than a compact conformation of TnC. In the binary TnC.TnI complex and the presence of Ca(2+), Met43 in TnC's N-domain was identified as the photo-cross-linking site, and multiple distances between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynamically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between the binary TnC.TnI and the ternary troponin complexes and the importance of using intact proteins in the study of structure-function relationships of troponin.
A recombinant soluble form of the human Fc gamma receptor was produced by engineering a cDNA construct containing the extracellular part of the mature protein. After expression in bacteria as inclusion body, the polypeptide was highly purified and was refolded in vitro with a method that was developed for the renaturation of immunoglobulin fragments. With this method oxidation of the disulfide bridges within the domains of the protein is done in the presence of an artificial 'chaperone' which protects the polypeptide molecules from unwanted protein protein interactions thereby inhibiting the incorrect oxidation of the SH-groups. and misfolding of the protein. The refolded recombinant soluble Fc gamma RIIb showed several characteristics of the native receptor: (i) it was recognized by a series of monoclonal antibodies specific for, and in most cases produced against the native cell-surface receptor: (ii) it is bound to its ligand (the Fc-region of different immunoglobulins) under very diverse conditions: and (iii) it is competed strongly and specifically with the native cell surface receptor for both ligand and antibody binding in experiments with distinct read-outs; (iv) monoclonal antibodies produced against the recombinant protein specifically recognized Fc gamma RIIb on different cells. From these data it was concluded that the recombinant soluble Fc-receptor was in a native, functionally active form, and its function was not affected by the lack of glycosylation.
The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.
BACKGROUND AND STUDY AIMS: Reporting and machine control based on speech technology can enhance work efficiency in the gastrointestinal endoscopy laboratory. MATERIALS AND METHODS: The status and activation of endoscopy laboratory equipment were described as a multivariate parameter and function system. Speech recognition, text evaluation and action definition engines were installed. Special programs were developed for the grammatical analysis of command sentences, and a rule-based expert system for the definition of machine answers. A speech backup engine provides feedback to the user. Techniques were applied based on the "Hidden Markov" model of discrete word, user-independent speech recognition and on phoneme-based speech synthesis. Speech samples were collected from three male low-tone investigators. RESULTS: The dictation module and machine control modules were incorporated in a personal computer (PC) simulation program. Altogether 100 unidentified patient records were analyzed. The sentences were grouped according to keywords, which indicate the main topics of a gastrointestinal endoscopy report. They were: "endoscope", "esophagus", "cardia", "fundus", "corpus", "antrum", "pylorus", "bulbus", and "postbulbar section", in addition to the major pathological findings: "erosion", "ulceration", and "malignancy". "Biopsy" and "diagnosis" were also included. We implemented wireless speech communication control commands for equipment including an endoscopy unit, video, monitor, printer, and PC. The recognition rate was 95%. CONCLUSIONS: Speech technology may soon become an integrated part of our daily routine in the endoscopy laboratory. A central speech and laboratory computer could be the most efficient alternative to having separate speech recognition units in all items of equipment.
Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.
It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644], and confirmed by structural studies [Gagne et al. (1995) Nat. Struct. Biol. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further events in the Ca2+ regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mutants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed by reconstitution and UV irradiation. Studies were made in the binary complex composed of TnC and TnI, the ternary complex composed of TnC, TnI, and troponin-T (TnT), and the synthetic thin filament composed of troponin, tropomyosin, and F-actin. TnC-TnI photo-cross-linking was observed for both mutants and for all three types of complexes. Although no Ca2+ dependence in the photo-cross-linking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2+ in the thin filament. TnI Met121, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC mutants using microsequencing and mass spectrometry following proteolysis. These results, obtained with intact TnC.TnI complexes, indicate that the TnI segment containing Met121 is in close contact with the N-terminal hydrophobic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of Ca2+, possibly triggering the translocation of the actin-binding region(s) of TnI toward actin.
ITIM-bearing receptors, a family which only recently has been recognized, play a key role in the regulation of the ITAM-induced activation of immune competent cells. The mechanism of ITM-mediated regulation in various cells was recently clarified. The present review focuses on ITIM bearing membrane proteins that negatively regulate the activation of cells when co-crosslinked with ITAM containing receptors, illustrates the inhibitory processes by the negative regulation of B-, NK-, T-cells and mast cells and summarizes current views on the mechanism of ITIM-mediated inhibition.
Co-clustering B cell receptors (BCR) and type II receptors binding the Fc part of IgG (Fc gamma RIIb) inhibits B cell activation and antibody production. Tyrosine phosphorylation of an intracellular motif of Fc gamma RIIb has been shown to be a prerequisite of the inhibition. After being phosphorylated by BCR-activated tyrosine kinases, the immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of Fc gamma RIIb recruits SH2 domain containing protein tyrosine phosphatase(s) (PTPs) and polyphosphoinositol 5-phosphatase (SHIP) to the vicinity of BCR, which in turn dephosphorylate their specific substrates. This leads to the interruption of signal transduction, consequently to the anergy and/or apoptosis of the cell. The downstream signaling pathways affected by Fc gamma RIIb-BCR co-clustering are not clarified yet, neither the substrates of PTPs are known. We have studied the Fc gamma RIIb mediated B cell inhibition on human Burkitt lymphoma cell line (BL41). From the lysates of BL41 cells SHP-2 and phosphatidylinositol 3-kinase (PI3-K), as well as the protein tyrosine kinase (PTK) Lyn bind both to the BCR-co-clustered Fc gamma RIIb and to its P-ITIM peptide. Lyn hyperphosphorylates the P-ITIM associated molecules, including SHIP in the in vitro protein tyrosine kinase activity assay. The P-ITIM-compelled multi-phosphoprotein complex binds to and activates SHP-2, which in turn dephosphorylates SHIP and Shc and probably other substrates. Subcellular localisation of these signaling molecules is regulated by the phosphotyrosine-SH2 domain interactions, thus dephosphorylation may result in the re-direction of Shc and SHIP within the cell, consequently, in the modulation of their activity. Finally, co-clustering Fc gamma RIIb and BCR or Fc gamma RIIb and CD19 on the intact cells inhibited PI3-K activity as detected in the anti-phosphotyrosine (anti-PY) precipitates. The results indicate that SHP-2 bound to and activated by the BCR co-clustered Fc gamma RIIb, may down-regulate PI3-K activity by dephosphorylating a yet unidentified regulatory molecule, which recruits PI3-K to the cell membrane.
Co-clustering of the type II receptors binding the Fc part of IgG (FcgammaRIIb) and B cell receptors results in the translocation of cytosolic, negative regulatory molecules to the phosphorylated immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of the FcgammaRIIb. SH2 domain-containing protein tyrosine phosphatases (SHP-1 and SHP-2), and the polyphosphoinositol 5'-phosphatase (SHIP) have been reported earlier to bind to murine FcgammaRIIb P-ITIM. However, neither the functional substrates of these enzymes, nor the mechanism of the inhibition are fully resolved. We show here that the human FcgammaRIIb binds SHP-2 when co-clustered with the B cell receptors, whereas its synthetic P-ITIM peptide bindes SHP-2 and SHIP in lysates of the Burkitt's lymphoma cell line BL41. The P-ITIM peptide binding enhances SHP-2 activity, resulting in dephosphorylation and release of P-ITIM-bound SHIP and Shc. Moreover, P-ITIM-bound SHP-2 dephosphorylates synthetic peptides corresponding to the sites of tyrosine phosphorylation on SHIP and Shc, indicating that these proteins are its potential substrates. Thus SHP-2-induced dephosphorylation may modulate the intracellular localization and/or activity of SHIP and Shc, thereby inhibiting further activation pathways which they mediate.
The field of striated muscle regulation has changed tremendously over the last forty years. Many of the problems solved by Dr. Ebashi and by those stimulated by him offer new challenges for future generations of scientists. Many questions remain to be solved, and it should give particular pleasure to Dr. Ebashi to see how the seeds sown by him and his colleagues have now grown into a beautiful tree that bears rich fruit at present and will continue to do so for a long time in the future.
Discrete, Hidden Markov model based speech recognition and phoneme based speech synthesis techniques were applied for gastroscopy reporting and machine control. The authors developed a special program for grammatical analysis of the sentences. Altogether 100 patient findings were grammatically analysed. The sentences were grouped according to the topographical order of the investigation: oesophagus, cardia, fundus, corpus, antrum, pylorus, bulbus, postbulbar section, and the pathological findings: erosion, ulceration, malignancy. Speech samples from 3 deep voiced male investigators were collected. The recognition rate was above 95%. A simulation program was also developed for dictation and controlling of the different equipment (monitor, printer, video, endoscope) in the gastroscopy laboratory by speech recognition. Speech synthesis was applied for the evaluation of understanding. This module artificially synthesizes the answer of the system giving backup for the understood information. With additional developments the discrete word speech 'recognition' achieved the level of routine application in medical reporting. However, ready-to-use developments need the joint activity of speech technology and endoscopy industry with end-user teams.
The contraction of vertebrate striated muscle contraction, and hence its work output, is controlled by Ca2+, which binds to troponin (Tn) associated with tropomyosin (TM) and actin in the thin filaments. Tn consists of three subunits: TnC, the Ca(2+)-receptor; TnI, an inhibitor of actomyosin activity; and TnT, anchoring Tn to TM. Of the four Ca(2+)-binding sites, I and II in the N-terminal domain are Ca-specific sites, while sites III and IV, the high affinity Ca-Mg sites, are in the C-domain. The former are recognized as the functionally important triggering sites. TnC, whose structure has been solved by X-ray crystallography and recently by high-resolution NMR, contains two homologous globular domains connected by an unusual single alpha-helix. The C-terminal domain exhibits an open hydrophobic area regardless of whether Ca2+ or Mg2+ is bound to sites III and IV. In contrast, the N-terminal domain is a closed structure that opens a hydrophobic patch upon Ca(2+)-binding to its two "triggering" sites producing a TnI binding area. Crosslinking and fragment binding studies indicate that, in the main, the two polypeptide chains run in opposite directions in the complex of TnC with Tn. A model of TnC-TnI interactions based on low angle X-ray and neutron scattering is discussed in light of biochemical and other physico-chemical studies. The opening of the structure in the N-terminal domain of TnC may be regarded as a molecular switch. It activates a molecular switch in TnI, reflected in the movement of portions of its C-terminal half, including Cys 133, away from actin and closer to TnC, as well as other structural changes in TnI. Finally the role of TnT in switching and transmitting the Ca(2+)-signal is discussed.
We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.
Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments of TnC and TnI. In this work we extended the mapping of the TnC-TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. The results are discussed in light of the antiparallel model of the TnI-TnC complex and a structural model derived from low-angle X-ray and neutron scattering studies.
Fc gammaRIIb (CD32) controls antibody production by down-regulating cell activation, when co-clustered with B cell antigen receptors (BCR) in vivo, via immune complexes consisting of secreted IgG and antigen. Fc gammaRIIb-BCR co-ligation in vitro was shown to inhibit the Ca2+ influx from the extracellular space, the mechanism of which is not fully understood. Human B cells express Fc gammaRIIb1 and Fc gammaRIIb2, differing only in a 19 amino acid long insert in the cytoplasmic tail of the former. To elucidate whether Fc gammaRIIb1 and Fc gammaRIIb2 isoforms show any difference in the down-regulation of B cells, we have studied the effect of co-clustering of BCR and Fc gammaRIIb1 or Fc gammaRIIb2 on the Ca2+ signaling in a Burkitt's lymphoma cell line, ST486, transfected with the two isoforms respectively. We have shown here, for the first time, that co-aggregation of BCR and Fc gammaRIIb may also inhibit Ca2+ release from the endoplasmic reticulum pool of human B cells. Both isoforms mediated this inhibition and the inhibitory effect depended on the ratio of BCR to Fc gammaRIIb cross-linking. In contrast to Fc gammaRIIb, the CD21/CD19 complex was shown to up-regulate B cell response by lowering the activation threshold. We have shown here that co-clustering of Fc gammaRIIb with CD19 inhibited the CD19-induced Ca2+ influx. Furthermore, the three party co-aggregation of Fc gammaRIIb with BCR and CD19 resulted in a decreased Ca2+ response, as compared to the BCR- plus CD19-induced one, indicating that Fc gammaRIIb may inhibit CD19-induced enhancement of B cell activation. On the basis of these data we suggest that IgG-containing and C3d-fixing immune complexes may down-regulate the B cell response by interfering with both BCR- and CD19-mediated Ca2+ mobilization.