scholarly journals Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox

2003 ◽  
Vol 374 (2) ◽  
pp. 433-441 ◽  
Author(s):  
Miki OKADA ◽  
Hideaki ITOH ◽  
Takashi HATAKEYAMA ◽  
Hiroshi TOKUMITSU ◽  
Ryoji KOBAYASHI
Keyword(s):  
Citrate Synthase ◽  
Heat Shock Protein 90 ◽  
Chaperone Activity ◽  
Physiological Significance ◽  
Disodium Cromoglycate ◽  
Terminal Domain ◽  
C Terminus

Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5–3.0 μM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.

scholarly journals Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

2007 ◽  
Vol 404 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Gary Flom ◽  
Robert H. Behal ◽  
Luke Rosen ◽  
Douglas G. Cole ◽  
Jill L. Johnson
Keyword(s):  
Current Model ◽  
Heat Shock Protein 90 ◽  
C Terminus ◽  
Client Proteins ◽  
Definition Of ◽  
Necessary And Sufficient ◽  
In Vitro Interaction

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.

scholarly journals DUK114, the Drosophila orthologue of bovine brain calpain activator protein, is a molecular chaperone

2004 ◽  
Vol 383 (1) ◽  
pp. 165-170 ◽  
Author(s):  
Attila FARKAS ◽  
Gábor NARDAI ◽  
Peter CSERMELY ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Heat Shock ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Citrate Synthase ◽  
Sequence Similarity ◽  
Heat Shock Protein 90 ◽  
Chaperone Activity ◽  
In Vitro Assays

UK114, the goat liver tumour antigen, is a member of a widely distributed family of conserved low-molecular-mass proteins (YER057c/YjgF/UK114), the function of which is ill understood. To the various orthologues diverse functions have been ascribed, such as translation inhibition, regulation of purine repressor or calpain activation. Owing to a limited sequence similarity to Hsp90 (heat-shock protein 90), they have also been proposed to be molecular chaperones; however, this has never been tested. In the present paper, we report the cloning and characterization of the Drosophila orthologue, DUK114. In brief, DUK114 had no effect that would have qualified it as a calpain activator. In contrast, it proved to be a very potent molecular chaperone in in vitro assays. In a heat-aggregation test, it significantly decelerated the formation of citrate synthase aggregates. In a reverse assay, the recovery of the enzyme from urea- and heat-induced denatured states was accelerated almost 3-fold. On a molar basis, the chaperone activity of the 15-kDa DUK114 is comparable with that of Hsp90, the almost 6-times-larger archetypal molecular chaperone. In similar assays, DUK114 was ineffective with Drosophila calpain A or calpain B. To test for its chaperone activity in vivo, DUK114 was transfected into Schneider (S2) cells; after heat shock, the number of viable non-transfected cells started to increase after a lag time; in the presence of DUK114, cell proliferation started at once. Our work is the first experimental evidence that DUK114, and possibly other members of this family, are molecular chaperones.

scholarly journals Functions of the C-Terminal Domain of Varicella-Zoster Virus Glycoprotein E in Viral Replication In Vitro and Skin and T-Cell Tropism In Vivo

2004 ◽  
Vol 78 (22) ◽  
pp. 12406-12415 ◽  
Author(s):  
Jennifer Moffat ◽  
Chengjun Mo ◽  
Jason J. Cheng ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
...  
Keyword(s):  
T Cell ◽  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Point Mutations ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an “acid cluster” comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.

scholarly journals Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo

2006 ◽  
Vol 17 (2) ◽  
pp. 907-916 ◽  
Author(s):  
Anne Straube ◽  
Gerd Hause ◽  
Gero Fink ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Activity ◽  
C Terminus ◽  
Cross Bridges ◽  
Conventional Kinesin ◽  
Motor Head

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals Regulation of Kif15 localization and motility by the C-terminus of TPX2 and microtubule dynamics

2017 ◽  
Vol 28 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Barbara J. Mann ◽  
Sai K. Balchand ◽  
Patricia Wadsworth
Keyword(s):  
Equatorial Region ◽  
Growth Reduction ◽  
Spindle Formation ◽  
Terminal Domain ◽  
C Terminus ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
In Cells

Mitotic motor proteins generate force to establish and maintain spindle bipolarity, but how they are temporally and spatially regulated in vivo is unclear. Prior work demonstrated that a microtubule-associated protein, TPX2, targets kinesin-5 and kinesin-12 motors to spindle microtubules. The C-terminal domain of TPX2 contributes to the localization and motility of the kinesin-5, Eg5, but it is not known whether this domain regulates kinesin-12, Kif15. We found that the C-terminal domain of TPX2 contributes to the localization of Kif15 to spindle microtubules in cells and suppresses motor walking in vitro. Kif15 and Eg5 are partially redundant motors, and overexpressed Kif15 can drive spindle formation in the absence of Eg5 activity. Kif15-dependent bipolar spindle formation in vivo requires the C-terminal domain of TPX2. In the spindle, fluorescent puncta of GFP-Kif15 move toward the equatorial region at a rate equivalent to microtubule growth. Reduction of microtubule growth with paclitaxel suppresses GFP-Kif15 motility, demonstrating that dynamic microtubules contribute to Kif15 behavior. Our results show that the C-terminal region of TPX2 regulates Kif15 in vitro, contributes to motor localization in cells, and is required for Kif15 force generation in vivo and further reveal that dynamic microtubules contribute to Kif15 behavior in vivo.

scholarly journals Twin-arginine translocase component TatB performs folding quality control via a general chaperone activity

2020 ◽  
Author(s):  
May N. Taw ◽  
Jason T. Boock ◽  
Daniel Kim ◽  
Mark A. Rocco ◽  
Dujduan Waraho-Zhmayev ◽  
...  
Keyword(s):  
Quality Control ◽  
Citrate Synthase ◽  
Active Species ◽  
Chaperone Activity ◽  
Twin Arginine Translocation ◽  
Substrate Protein ◽  
Twin Arginine Translocase ◽  
Partially Unfolded

AbstractThe twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) system that synchronizes proofreading of substrate protein folding with lipid bilayer transport. However, the molecular details of this QC mechanism remain poorly understood. Here, we hypothesized that the conformational state of Tat substrates is directly sensed by the TatB component of the bacterial Tat translocase. In support of this hypothesis, several TatB variants in which the cytoplasmic membrane-extrinsic domain was either truncated or mutated in the vicinity of a conserved, highly flexible α-helical domain were observed to form functional translocases in vivo that had compromised QC activity as evidenced by the uncharacteristic export of several misfolded protein substrates. In vitro folding experiments revealed that the membrane-extrinsic domain of TatB possessed general chaperone activity, transiently binding to highly structured, partially unfolded intermediates of a model protein, citrate synthase, thereby preventing its irreversible aggregation and stabilizing the active species. Collectively, these results suggest that the Tat translocase may use chaperone-like client recognition to monitor the conformational status of its substrates.

scholarly journals C-Terminal Domain of ABL Family Kinases, ABL and ARG, Defines Their Distinct Leukemogenic Activities in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2368-2368 ◽  
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Tsukimi Shoji ◽  
Taira Maekawa ◽  
Keiko Okuda
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Fusion Proteins ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Terminal Domain ◽  
C Terminus

Abstract ARG (ABL2) is a member of ABL family kinases and highly homologous to ABL (ABL1) except the C-terminal domain adjacent to the kinase domain. TEL/ARG that consists of ARG fused to TEL (ETV6) has been found in AML M3, M4 or T-ALL patients, with additional chromosomal abnormalities of t(15;17)(q12;q21), inv(16)(p13;q12) or t(1;10;12)(q25;q23;p13) translocation, respectively. The structure of TEL/ARG is similar to that of TEL/ABL, which has been found in patients with T-ALL, B-ALL, AML and CML. TEL mediates homo-oligomerization of these fusion proteins, TEL/ABL and TEL/ARG, resulting in constitutive activation of the tyrosine kinases. Although ABL fusion proteins such as BCR/ABL and TEL/ABL have been intensively investigated, the involvement of TEL/ARG in leukemogenesis is not fully elucidated yet. We have recently reported that in vitro transforming activity of TEL/ARG was significantly lower than that of TEL/ABL although their kinase activities were almost identical. Interestingly, the in vitro transforming activities of C-terminus-swapped mutants, TEL/ABL with C-terminal domain of ARG [TEL-ABL (ARG-C)] or TEL/ARG with C-terminal domain of ABL [TEL/ARG (ABL-C)], were comparable to those of TEL/ARG or TEL/ABL, respectively, while kinase activities in the swapped mutants were not altered. These results suggest that C-termini of ABL family kinases contain some functional domain that defines their distinct transforming activities. The purpose of this study is to compare the in vivo leukemogenic activities of TEL/ABL and TEL/ARG, and evaluate the impact of the C-terminal domains. First, we investigated whether TEL/ABL or TEL/ARG caused leukemia in mice. Each fusion gene together with GFP gene was retrovirally transduced into the bone marrow cells harvested from C57BL/6 mice treated with 5-fluorouracil, and the transduced cells were transplanted into lethally irradiated mice. Similar to BCR/ABL, transplantation of TEL/ABL-transduced cells induced rapid myeloproliferative status accompanied by hepatomegaly and/or splenomegaly, and all the recipient mice died within 33 days after transplantation, indicating the development of myeloid leukemia. In contrast, the recipient mice transplanted with TEL/ARG-transduced cells did not develop myeloid leukemia but infiltrative mastocytosis, and died around 200 days after transplantation (Figure 1). Hemophagocytic mast cells accumulating in the bone marrow, and mast cells circulating in the peripheral blood were also observed in these mice. Next we investigated the roles of C-terminal domains of ABL and ARG in their in vivo leukemogenic activities. C-terminus-swapped mutants, TEL/ABL (ARG-C) and TEL/ARG (ABL-C) were retrovirally transduced into bone marrow cells and the transduced cells were transplanted as described above. Intriguingly, TEL/ABL (ARG-C) mutant failed to cause myeloproliferative status or leukemia at day 153 (Figure 2A). On the other hand, TEL/ARG (ABL-C) induced lethal myeloid leukemia in 4 out of 13 mice (30.8%) within 111 days after transplantation (Figure 2B). Collectively, the in vivo phenotypes induced by TEL/ABL (ARG-C) or TEL/ARG (ABL-C) resembled those induced by TEL/ARG or TEL/ABL, respectively. Mastocytosis, a characteristic of TEL-ARG-induced phenotype, has not been observed so far in any of the recipients of TEL/ABL (ARG-C) or TEL/ARG (ABL-C). In conclusion, these results indicate that C-terminal domain of ABL family kinases defines their distinct leukemogenic activities in vivo through modulating both proliferation and differentiation. Notably, C-terminus of ARG strongly suppressed the in vivo leukemogenic activity of TEL/ABL without impairing the tyrosine kinase activity. Further clarification of the molecular mechanisms underlying the suppressive activity of C-terminus of ARG will lead to development of a novel therapeutic strategy, especially for patients with CML harboring mutations, which are resistant to tyrosine kinase inhibitors. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals RFWD2 Knockdown as a Blocker to Reverse the Oncogenic Role of TRIB2 in Lung Adenocarcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Ruimin Hao ◽  
Jinxia Hu ◽  
Yuemei Liu ◽  
Dongmin Liang ◽  
Yan-Mei Li ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Cancer Cell Proliferation ◽  
Cancer Cell Growth ◽  
Specific Domain ◽  
Cancer Breast ◽  
C Terminus ◽  
And Migration

RFWD2, an E3 ubiquitin ligase, is overexpressed in numerous human cancers, including leukemia, lung cancer, breast cancer, renal cell carcinoma, and colorectal cancer. The roles of RFWD2 in cancer are related to the targeting of its substrates for ubiquitination and degradation. This study aimed to investigate the role of TRIB2 in relation to the regulation of protein degradation through RFWD2. inBio Discover™ results demonstrated that TRIB2 can perform its functions by interacting with RFWD2 or other factors. TRIB2 can interact with and regulate RFWD2, which further attends the proteasome-mediated degradation of the RFWD2 substrate p-IκB-α. TRIB2 colocalizes with RFWD2-related IκB-α to form a ternary complex and further affects the IκB-α degradation by regulating its phosphorylation. Specific domain analysis showed that TRIB2 may bind to RFWD2 via its C-terminus, whereas it binds to IκB via its pseudokinase domain. TRIB2 acts as an oncogene and promotes cancer cell proliferation and migration, whereas RFWD2 knockdown reversed the role of TRIB2 in promoting cancer cell growth and colony formation in vitro and in vivo. In summary, this study reveals that TRIB2 promotes the progression of cancer by affecting the proteasome-mediated degradation of proteins through the interaction with RFWD2.

scholarly journals Role of the C-Terminal Domain of the Alpha Subunit of RNA Polymerase in LuxR-Dependent Transcriptional Activation of the lux Operon during Quorum Sensing

2002 ◽  
Vol 184 (16) ◽  
pp. 4520-4528 ◽  
Author(s):  
Angela H. Finney ◽  
Robert J. Blick ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Α Subunit ◽  
Terminal Domain

ABSTRACT During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [N-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the lux operon promoter at a position centered at −42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the α-subunit C-terminal domain (αCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effects of 70 alanine substitution variants of the α subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRΔN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRΔN, and 14 alanine substitutions in the αCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these 14 α variants were also involved in the mechanisms of both LuxR- and LuxRΔN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in α that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in α play roles in DNA recognition and residues 290 and 314 play roles in α-LuxR interactions at the lux operon promoter during quorum sensing.

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